Type VI CRISPR orthologs and systems

ABSTRACT

The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered RNA-targeting systems comprising a novel RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to U.S. Provisional 62/351,662 and62/351,803, filed on Jun. 17, 2016, U.S. Provisional 62/376,377, filedon Aug. 17, 2016, U.S. Provisional 62/410,366, filed Oct. 19, 2016, U.S.Provisional 62/432,240, filed Dec. 9, 2016, U.S. provisional 62/471,792filed Mar. 15, 2017, and U.S. Provisional 62/484,786 filed Apr. 12,2017.

Reference is made to U.S. Provisional 62/471,710, filed Mar. 15, 2017(entitled, “Novel Cas13B Orthologues CRISPR Enzymes and Systems,”.Reference is further made to U.S. Provisional 62/432,553, filed Dec. 9,2016, U.S. Provisional 62/456,645, filed Feb. 8, 2017, and U.S.Provisional 62/471,930, filed Mar. 15, 2017 (entitled “CRISPR EffectorSystem Based Diagnostics,” and U.S. Provisional 62/484,869, filed Apr.12, 2017 (entitled “CRISPR Effector System Based Diagnostics,”.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersMH100706 and MH110049 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

All documents cited or referenced in herein cited documents, togetherwith any manufacturer's instructions, descriptions, productspecifications, and product sheets for any products mentioned herein orin any document incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. More specifically, all referenced documents areincorporated by reference to the same extent as if each individualdocument was specifically and individually indicated to be incorporatedby reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (“BROD-2710US_ST25.txt”;Size is 1,357,511 bytes and it was created on May 3, 2022) is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to systems, methods andcompositions used for the control of gene expression involving sequencetargeting, such as perturbation of gene transcripts or nucleic acidediting, that may use vector systems related to Clustered RegularlyInterspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND OF THE INVENTION

Recent advances in genome sequencing techniques and analysis methodshave significantly accelerated the ability to catalog and map geneticfactors associated with a diverse range of biological functions anddiseases. Precise genome targeting technologies are needed to enablesystematic reverse engineering of causal genetic variations by allowingselective perturbation of individual genetic elements, as well as toadvance synthetic biology, biotechnological, and medical applications.Although genome-editing techniques such as designer zinc fingers,transcription activator-like effectors (TALEs), or homing meganucleasesare available for producing targeted genome perturbations, there remainsa need for new genome and transcriptome engineering technologies thatemploy novel strategies and molecular mechanisms and are affordable,easy to set up, scalable, and amenable to targeting multiple positionswithin the eukaryotic genome and transcriptome. This would provide amajor resource for new applications in genome engineering andbiotechnology.

The CRISPR-Cas systems of bacterial and archaeal adaptive immunity showextreme diversity of protein composition and genomic loci architecture.The CRISPR-Cas system loci has more than 50 gene families and there isno strictly universal genes indicating fast evolution and extremediversity of loci architecture. So far, adopting a multi-prongedapproach, there is comprehensive cas gene identification of about 395profiles for 93 Cas proteins. Classification includes signature geneprofiles plus signatures of locus architecture. A new classification ofCRISPR-Cas systems is proposed in which these systems are broadlydivided into two classes, Class 1 with multisubunit effector complexesand Class 2 with single-subunit effector modules exemplified by the Cas9protein. Novel effector proteins associated with Class 2 CRISPR-Cassystems may be developed as powerful genome engineering tools and theprediction of putative novel effector proteins and their engineering andoptimization is important.

The CRISPR-Cas adaptive immune system defends microbes against foreigngenetic elements via DNA or RNA-DNA interference. Recently, the Class 2type VI single-component CRISPR-Cas effector C2c2 (Shmakov et al. (2015)“Discovery and Functional Characterization of Diverse Class 2 CRISPR-CasSystems”; Molecular Cell 60:1-13; doi:http://dx.doi.org/10.1016/j.molcel.2015.10.008) was characterized as anRNA-guided Rnase (Abudayyeh et al. (2016), Science, [Epub ahead ofprint], June 2; “C2c2 is a single-component programmable RNA-guidedRNA-targeting CRISPR effector”; doi: 10.1126/science.aaf5573). It wasdemonstrated that C2c2 (e.g. from Leptotrichia shahii) provides robustinterference against RNA phage infection. Through in vitro biochemicalanalysis and in vivo assays, it was shown that C2c2 can be programmed tocleave ssRNA targets carrying protospacers flanked by a 3′ H (non-G)PAM. Cleavage is mediated by catalytic residues in the two conservedHEPN domains of C2c2, mutations in which generate a catalyticallyinactive RNA-binding protein. C2c2 is guided by a single guide and canbe re-programmed to deplete specific mRNAs in vivo. It was shown thatLshC2c2 can be targeted to a specific site of interest and can carry outnon-specific RNase activity once primed with the cognate target RNA.These results broaden our understanding of CRISPR-Cas systems anddemonstrate the possibility of harnessing C2c2 to develop a broad set ofRNA-targeting tools.

C2c2 is now known as Cas13a. It will be understood that the term “C2c2”herein is used interchangeably with “Cas13a”.

Citation or identification of any document in this application is not anadmission that such document is available as prior art to the presentinvention.

SUMMARY OF THE INVENTION

There exists a pressing need for alternative and robust systems andtechniques for targeting nucleic acids or polynucleotides (e.g. DNA orRNA or any hybrid or derivative thereof) with a wide array ofapplications, in particular in eukaryotic systems, more in particular inmammalian systems. This invention addresses this need and providesrelated advantages. Adding the novel RNA-targeting systems of thepresent application to the repertoire of genomic, transcriptomic, andepigenomic targeting technologies may transform the study andperturbation or editing of specific target sites through directdetection, analysis and manipulation, in particular in eukaryoticsystems, more in particular in mammalian systems (including cells,organs, tissues, or organisms) and plant systems. To utilize theRNA-targeting systems of the present application effectively for RNAtargeting without deleterious effects, it is critical to understandaspects of engineering and optimization of these RNA targeting tools.

The CRISPR-Cas13 family was discovered by computational mining ofbacterial genomes for signatures of CRISPR systems (Shmakov, S. et al.Discovery and Functional Characterization of Diverse Class 2 CRISPR-CasSystems. Mol Cell 60, 385-397, doi:10.1016/j.molcel.2015.10.008 (2015)),revealing the single-effector RNA-guided RNase Cas13a/C2c2 (Abudayyeh,O. O. et al. C2c2 is a single-component programmable RNA-guidedRNA-targeting CRISPR effector. Science 353, aaf5573,doi:10.1126/science.aaf5573 (2016)) and later the single-effectorRNA-guided RNase Cas13b (Shmakov, S. et al. Diversity and evolution ofclass 2 CRISPR-Cas systems. Nat Rev Microbiol 15, 169-182,doi:10.1038/nrmicro.2016.184 (2017); Smargon, A. A. et al. Cas13b Is aType VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated byAccessory Proteins Csx27 and Csx28. Mol Cell 65, 618-630 e617,doi:10.1016/j.molcel.2016.12.023 (2017)). The Class 2 type VI effectorprotein C2c2, also known as Cas13a, is a RNA-guided RNase that can beefficiently programmed to degrade ssRNA. C2c2 (Cas13a) achieves RNAcleavage through conserved basic residues within its two HEPN domains,in contrast to the catalytic mechanisms of other known RNases found inCRISPR-Cas systems. Mutation of the HEPN domain, such as (e.g. alanine)substitution, at any of the four predicted HEPN domain catalyticresidues converted C2c2 into an inactive programmable RNA-bindingprotein (dC2c2, analogous to dCas9).

The programmability and specificity of the RNA-guided RNase Cas13 wouldmake it an ideal platform for transcriptome manipulation. Applicantsdevelop Cas13a for use as a mammalian transcript knockdown and bindingtool. Cas13a from Leptotrichia shahii (LshCas13a) is capable of robustRNA cleavage and binding with catalytically inactive versions usingprogrammable crRNAs and that cleavage was dependent on a directly3′-adjacent motif known as the protospacer flanking site (PFS) withidentity H (not guanine) (Abudayyeh, O. O. et al. C2c2 is asingle-component programmable RNA-guided RNA-targeting CRISPR effector.Science 353, aaf5573, doi:10.1126/science.aaf5573 (2016)). Upon RNAcleavage, activated LshCas13a engages in “collateral activity” in whichconstitutive RNase activity cleaves non-targeted RNAs (Abudayyeh, O. O.et al. C2c2 is a single-component programmable RNA-guided RNA-targetingCRISPR effector. Science 353, aaf5573, doi:10.1126/science.aaf5573(2016)). This crRNA-programmed collateral activity enables in vivoprogrammed cell death by the bacteria to prevent spread of infection(Abudayyeh, O. O. et al. C2c2 is a single-component programmableRNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573,doi:10.1126/science.aaf5573 (2016)) and has been applied in vitro forthe specific detection of nucleic acid (Abudayyeh, O. O. et al. C2c2 isa single-component programmable RNA-guided RNA-targeting CRISPReffector. Science 353, aaf5573, doi:10.1126/science.aaf5573 (2016);East-Seletsky, A. et al. Two distinct RNase activities of CRISPR-C2c2enable guide-RNA processing and RNA detection. Nature 538, 270-273,doi:10.1038/nature19802 (2016)). Collateral activity was recentlyleveraged for a highly sensitive and specific nucleic acid detectionplatform termed SHERLOCK that is useful for many clinical diagnoses(Gootenberg, J. S. et al. Nucleic acid detection withCRISPR-Cas13a/C2c2. Science 356, 438-442 (2017)).

Via screening Cas13a orthologs in bacterial and subsequent biochemicalcharacterization, Applicants select an ortholog optimized for RNAendonuclease activity, the Cas13a from Leptotrichia wadei (LwaCas13a).LwaCas13a can be stably expressed in mammalian cells, retargeted toeffectively knockdown both reporter and endogenous transcripts in cells,and attains levels of high levels of targeting specificity compared toRNAi without observable collateral activity. Furthermore, Applicantsshow that catalytically inactive LwaCas13a (dCas13a) programmably bindsRNA transcripts in vivo and can be used to image transcripts in cells.By engineering a negative-feedback imaging system based upon dCas13a,the formation of stress granules can be tracked in living cells.

The ability of dC2c2 (dCas13a) to bind to specified sequences could beused in several aspects according to the invention to (i) bring effectormodules to specific transcripts to modulate the function or translation,which could be used for large-scale screening, construction of syntheticregulatory circuits and other purposes; (ii) fluorescently tag specificRNAs to visualize their trafficking and/or localization; (iii) alter RNAlocalization through domains with affinity for specific subcellularcompartments; and (iv) capture specific transcripts (through direct pulldown of dC2c2 or use of dC2c2 to localize biotin ligase activity tospecific transcripts) to enrich for proximal molecular partners,including RNAs and proteins.

Active C2c2 should also have many applications. An aspect of theinvention involves targeting a specific transcript for destruction, aswith RFP here. In addition, C2c2, once primed by the cognate target, cancleave other (non-complementary) RNA molecules in vitro and can inhibitcell growth in vivo. Biologically, this promiscuous RNase activity mayreflect a programmed cell death/dormancy (PCD/D)-based protectionmechanism of the type VI CRISPR-Cas systems. Accordingly, in an aspectof the invention, it might be used to trigger PCD or dormancy inspecific cells—for example, cancer cells expressing a particulartranscript, neurons of a given class, cells infected by a specificpathogen, or other aberrant cells or cells the presence of which isotherwise undesirable.

The invention provides a method of modifying nucleic acid sequencesassociated with or at a target locus of interest, in particular ineukaryotic cells, tissues, organs, or organisms, more in particular inmammalian cells, tissues, organs, or organisms, the method comprisingdelivering to said locus a non-naturally occurring or engineeredcomposition comprising a Type VI CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the sequences associated with or at thetarget locus of interest. In a preferred embodiment, the modification isthe introduction of a strand break. In a preferred embodiment, thesequences associated with or at the target locus of interest comprisesRNA and the effector protein is encoded by a type VI CRISPR-Cas loci.The complex can be formed in vitro or ex vivo and introduced into a cellor contacted with RNA; or can be formed in vivo.

It will be appreciated that the terms Cas enzyme, CRISPR enzyme, CRISPRprotein Cas protein and CRISPR Cas are generally used interchangeablyand at all points of reference herein refer by analogy to novel CRISPReffector proteins further described in this application, unlessotherwise apparent, such as by specific reference to Cas9. The CRISPReffector proteins described herein are preferably C2c2 effectorproteins.

The invention provides a method of targeting (such as modifying)sequences associated with or at a target locus of interest, the methodcomprising delivering to said sequences associated with or at the locusa non-naturally occurring or engineered composition comprising a C2c2loci effector protein (which may be catalytically active, oralternatively catalytically inactive) and one or more nucleic acidcomponents, wherein the C2c2 effector protein forms a complex with theone or more nucleic acid components and upon binding of the said complexto the locus of interest the effector protein induces the modificationof sequences associated with or at the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak. In a preferred embodiment the C2c2 effector protein forms acomplex with one nucleic acid component; advantageously an engineered ornon-naturally occurring nucleic acid component. The complex can beformed in vitro or ex vivo and introduced into a cell or contacted withRNA; or can be formed in vivo. The induction of modification ofsequences associated with or at the target locus of interest can be C2c2effector protein-nucleic acid guided. In a preferred embodiment the onenucleic acid component is a CRISPR RNA (crRNA). In a preferredembodiment the one nucleic acid component is a mature crRNA or guideRNA, wherein the mature crRNA or guide RNA comprises a spacer sequence(or guide sequence) and a direct repeat sequence or derivatives thereof.In a preferred embodiment the spacer sequence or the derivative thereofcomprises a seed sequence, wherein the seed sequence is critical forrecognition and/or hybridization to the sequence at the target locus.

Aspects of the invention relate to C2c2 effector protein complexeshaving one or more non-naturally occurring or engineered or modified oroptimized nucleic acid components. In a preferred embodiment the nucleicacid component of the complex may comprise a guide sequence linked to adirect repeat sequence, wherein the direct repeat sequence comprises oneor more stem loops or optimized secondary structures. In certainembodiments, the direct repeat has a minimum length of 16 nts, such asat least 28 nt, and a single stem loop. In further embodiments thedirect repeat has a length longer than 16 nts, preferably more than 17nts, such as at least 28 nt, and has more than one stem loop oroptimized secondary structures. In particular embodiments, the directrepeat has 25 or more nts, such as 26 nt, 27 nt, 28 nt or more, and oneor more stem loop structures. In a preferred embodiment the directrepeat may be modified to comprise one or more protein-binding RNAaptamers. In a preferred embodiment, one or more aptamers may beincluded such as part of optimized secondary structure. Such aptamersmay be capable of binding a bacteriophage coat protein. Thebacteriophage coat protein may be selected from the group comprising Qβ,F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1,TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r,7s and PRR1. In a preferred embodiment the bacteriophage coat protein isMS2. The invention also provides for the nucleic acid component of thecomplex being 30 or more, 40 or more or 50 or more nucleotides inlength.

The invention provides cells comprising the type VI effector proteinand/or guides and/or complexes thereof with target nucleic acids. Incertain embodiments, the cell is a eukaryotic cell, including but notlimited to a yeast cell, a plant cell, a mammalian cell, an animal cell,or a human cell.

The invention also provides a method of modifying a target locus ofinterest, in particular in eukaryotic cells, tissues, organs, ororganisms, more in particular in mammalian cells, tissues, organs, ororganisms, the method comprising delivering to said locus anon-naturally occurring or engineered composition comprising a C2c2 locieffector protein and one or more nucleic acid components, wherein theC2c2 effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break. The complex can be formed in vitro or exvivo and introduced into a cell or contacted with RNA; or can be formedin vivo.

In such methods the target locus of interest may be comprised within anRNA molecule. Also, the target locus of interest may be comprised withina DNA molecule, and in certain embodiments, within a transcribed DNAmolecule. In such methods the target locus of interest may be comprisedin a nucleic acid molecule in vitro.

In such methods the target locus of interest may be comprised in anucleic acid molecule within a cell, in particular a eukaryotic cell,such as a mammalian cell or a plant cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Theplant cell may be of a crop plant such as cassava, corn, sorghum, wheat,or rice. The plant cell may also be of an algae, tree or vegetable. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell are altered for improvedproduction of biologic products such as an antibody, starch, alcohol orother desired cellular output. The modification introduced to the cellby the present invention may be such that the cell and progeny of thecell include an alteration that changes the biologic product produced.

The mammalian cell many be a non-human mammal, e.g., primate, bovine,ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep,pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalianeukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish(e.g., salmon) or shellfish (e.g., oyster, claim, lobster, shrimp) cell.The cell may also be a plant cell. The plant cell may be of a monocot ordicot or of a crop or grain plant such as cassava, corn, sorghum,soybean, wheat, oat or rice. The plant cell may also be of an algae,tree or production plant, fruit or vegetable (e.g., trees such as citrustrees, e.g., orange, grapefruit or lemon trees; peach or nectarinetrees; apple or pear trees; nut trees such as almond or walnut orpistachio trees; nightshade plants; plants of the genus Brassica; plantsof the genus Lactuca; plants of the genus Spinacia; plants of the genusCapsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli,cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry,blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

The invention provides a method of modifying a target locus of interest,the method comprising delivering to said locus a non-naturally occurringor engineered composition comprising a Type VI CRISPR-Cas loci effectorprotein and one or more nucleic acid components, wherein the effectorprotein forms a complex with the one or more nucleic acid components andupon binding of the said complex to the locus of interest the effectorprotein induces the modification of the target locus of interest. In apreferred embodiment, the modification is the introduction of a strandbreak.

The invention also provides a method of modifying a target locus ofinterest, the method comprising delivering to said locus a non-naturallyoccurring or engineered composition comprising a C2c2 loci effectorprotein and one or more nucleic acid components, wherein the C2c2effector protein forms a complex with the one or more nucleic acidcomponents and upon binding of the said complex to the locus of interestthe effector protein induces the modification of the target locus ofinterest. In a preferred embodiment, the modification is theintroduction of a strand break.

In such methods the target locus of interest may be comprised in anucleic acid molecule in vitro. In such methods the target locus ofinterest may be comprised in a nucleic acid molecule within a cell.Preferably, in such methods the target locus of interest may becomprised in a RNA molecule in vitro. Also preferably, in such methodsthe target locus of interest may be comprised in a RNA molecule within acell. The cell may be a prokaryotic cell or a eukaryotic cell. The cellmay be a mammalian cell. The cell may be a rodent cell. The cell may bea mouse cell.

In any of the described methods the target locus of interest may be agenomic or epigenomic locus of interest. In any of the described methodsthe complex may be delivered with multiple guides for multiplexed use.In any of the described methods more than one protein(s) may be used.

In further aspects of the invention the nucleic acid components maycomprise a CRISPR RNA (crRNA) sequence. Without limitation, theApplicants hypothesize that in such instances, the pre-crRNA maycomprise secondary structure that is sufficient for processing to yieldthe mature crRNA as well as crRNA loading onto the effector protein. Bymeans of example and not limitation, such secondary structure maycomprise, consist essentially of or consist of a stem loop within thepre-crRNA, more particularly within the direct repeat.

In any of the described methods the effector protein and nucleic acidcomponents may be provided via one or more polynucleotide moleculesencoding the protein and/or nucleic acid component(s), and wherein theone or more polynucleotide molecules are operably configured to expressthe protein and/or the nucleic acid component(s). The one or morepolynucleotide molecules may comprise one or more regulatory elementsoperably configured to express the protein and/or the nucleic acidcomponent(s). The one or more polynucleotide molecules may be comprisedwithin one or more vectors. In any of the described methods the targetlocus of interest may be a genomic or epigenomic locus of interest. Inany of the described methods the complex may be delivered with multipleguides for multiplexed use. In any of the described methods more thanone protein(s) may be used.

Regulatory elements may comprise inducible promotors. Polynucleotidesand/or vector systems may comprise inducible systems.

In any of the described methods the one or more polynucleotide moleculesmay be comprised in a delivery system, or the one or more vectors may becomprised in a delivery system.

In any of the described methods the non-naturally occurring orengineered composition may be delivered via liposomes, particlesincluding nanoparticles, exosomes, microvesicles, a gene-gun or one ormore viral vectors.

The invention also provides a non-naturally occurring or engineeredcomposition which is a composition having the characteristics asdiscussed herein or defined in any of the herein described methods.

In certain embodiments, the invention thus provides a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising a Type VI CRISPR-Cas loci effector protein andone or more nucleic acid components, wherein the effector protein formsa complex with the one or more nucleic acid components and upon bindingof the said complex to the locus of interest the effector proteininduces the modification of the target locus of interest. In certainembodiments, the effector protein may be a C2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition, such as particularly a compositioncapable of or configured to modify a target locus of interest, saidcomposition comprising: (a) a guide RNA molecule (or a combination ofguide RNA molecules, e.g., a first guide RNA molecule and a second guideRNA molecule, such as for multiplexing) or a nucleic acid encoding theguide RNA molecule (or one or more nucleic acids encoding thecombination of guide RNA molecules); (b) a Type VI CRISPR-Cas locieffector protein or a nucleic acid encoding the Type VI CRISPR-Cas locieffector protein. In certain embodiments, the effector protein may be aC2c2 loci effector protein.

The invention also provides in a further aspect a non-naturallyoccurring or engineered composition comprising: (a) a guide RNA molecule(or a combination of guide RNA molecules, e.g., a first guide RNAmolecule and a second guide RNA molecule) or a nucleic acid encoding theguide RNA molecule (or one or more nucleic acids encoding thecombination of guide RNA molecules); (b) be a C2c2 loci effectorprotein.

The invention also provides a vector system comprising one or morevectors, the one or more vectors comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics as definedin any of the herein described methods.

The invention also provides a delivery system comprising one or morevectors or one or more polynucleotide molecules, the one or more vectorsor polynucleotide molecules comprising one or more polynucleotidemolecules encoding components of a non-naturally occurring or engineeredcomposition which is a composition having the characteristics discussedherein or as defined in any of the herein described methods.

The invention also provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in atherapeutic method of treatment. The therapeutic method of treatment maycomprise gene or transcriptome editing, or gene therapy.

The invention also provides for methods and compositions wherein one ormore amino acid residues of the effector protein may be modified e.g.,an engineered or non-naturally-occurring effector protein or C2c2. In anembodiment, the modification may comprise mutation of one or more aminoacid residues of the effector protein. The one or more mutations may bein one or more catalytically active domains of the effector protein. Theeffector protein may have reduced or abolished nuclease activitycompared with an effector protein lacking said one or more mutations.The effector protein may not direct cleavage of the RNA strand at thetarget locus of interest. In a preferred embodiment, the one or moremutations may comprise two mutations. In a preferred embodiment the oneor more amino acid residues are modified in a C2c2 effector protein,e.g., an engineered or non-naturally-occurring effector protein or C2c2.In particular embodiments, the one or more modified or mutated aminoacid residues are one or more of those in C2c2 corresponding to R597,H602, R1278 and H1283 (referenced to LshC2c2 amino acids), such asmutations R597A, H602A, R1278A and H1283A, or the corresponding aminoacid residues in LshC2c2 orthologues.

In particular embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,V40, E479, L514, V518, N524, G534, K535, E580, L597, V602, D630, F676,L709, 1713, R717 (HEPN), N718, H722 (HEPN), E773, P823, V828, 1879,Y880, F884, Y997, L1001, F1009, L1013, Y1093, L1099, L1111, Y1114,L1203, D1222, Y1244, L1250, L1253, K1261, I1334, L1355, L1359, R1362,Y1366, E1371, R1372, D1373, R1509 (HEPN), H1514 (HEPN), Y1543, D1544,K1546, K1548, V1551, I1558, according to C2c2 consensus numbering. Incertain embodiments, the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to R717 andR1509. In certain embodiments, the one or more modified of mutated aminoacid residues are one or more of those in C2c2 corresponding to K2, K39,K535, K1261, R1362, R1372, K1546 and K1548. In certain embodiments, saidmutations result in a protein having an altered or modified activity. Incertain embodiments, said mutations result in a protein having anincreased activity, such as an increased specificity. In certainembodiments, said mutations result in a protein having a reducedactivity, such as reduced specificity. In certain embodiments, saidmutations result in a protein having no catalytic activity (i.e. “dead”C2c2). In an embodiment, said amino acid residues correspond to LshC2c2amino acid residues, or the corresponding amino acid residues of a C2c2protein from a different species.

In certain embodiments the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to M35, K36,T38, K39, 157, E65, G66, L68, N84, T86, E88, I103, N105, E123, R128,R129, K139, L152, L194, N196, K198, N201, Y222, D253, I266, F267, S280,I303, N306, R331, Y338, K389, Y390, K391, I434, K435, L458, D459, E462,L463, I478, E479, K494, R495, N498, S501, E519, N524, Y529, V530, G534,K535, Y539, T549, D551, R577, E580, A581, F582, I587, A593, L597, I601,L602, E611, E613, D630, I631, G633, K641, N646, V669, F676, S678, N695,E703, A707, I709, I713, I716, R717, H722, F740, F742, K768, I774, K778,I783, L787, S789, V792, Y796, D799, F812, N818, P820, F821, V822, P823,S824, F825, Y829, K831, D837, L852, F858, E867, A871, L875, K877, Y880,Y881, F884, F888, F896, N901, V903, N915, K916, R918, Q920, E951, P956,Y959, Q964, I969, N994, F1000, I10001, Q1003, F10005, K1007, G1008,F1009, N1019, L1020, K1021, I1023, N1028, E1070, I1075, K1076, F1092,K1097, L1099, L1104, L1107, K1113, Y1114, E1149, E1151, I1153, L1155,L1158, D1166, L1203, D1222, G1224, I1228, R1236, K1243, Y1244, G1245,D1255, K1261, S1263, L1267, E1269, K1274, I1277, E1278, L1289, H1290,A1294, N1320, K1325, E1327, Y1328, I1334, Y1337, K1341, N1342, K1343,N1350, L1352, L1355, L1356, I1359, L1360, R1362, V1363, G1364, Y1365,I1369, R1371, D1372, F1385, E1391, D1459, K1463, K1466, R1509, N1510,I1512, A1513, H1514, N1516, Y1517, L1529, L1530, E1534, L1536, R1537,Y1543, D1544, R1545, K1546, L1547, K1548, N1549, A1550, K1553, S1554,D1557, I1558, L1559, G1563, F1568, I1612, L1651, E1652, K1655, H1658,L1659, K1663, T1673, S1677, E1678, E1679, C1681, V1684, K1685, E1689with reference to the consensus sequence as indicated in FIG. 3 , i.e.based on the alignment of Leptotrichia wadei F0279 (“Lew2” or “Lw2”) andListeria newyorkensis FSL M6-0635 (also known as Listeriaceae bacteriumFSL M6-0635 (“Lib” or “LbFSL”)). As indicated earlier, in certainembodiments, in the above amino acid residue list, the residuescorresponding to R597, H602, R1278 and H1283 (referenced to LshC2c2amino acids) are excluded.

In certain embodiments, the one or more modified of mutated amino acidresidues are one or more conserved charged amino acid residues. Incertain embodiments, said amino acid residues may be mutated to alanine.

In certain embodiments the one or more modified of mutated amino acidresidues are one or more of those in C2c2 corresponding to K28, K31,R44, E162, E184, K262, E288, K357, E360, K338, R441 (HEPN), H446 (HEPN),E471, K482, K525, K558, D707, R790, K811, R833, E839, R885, E894, R895,D896, K942, R960 (HEPN), H965 (HEPN), D990, K992, K994 with reference tothe consensus sequence as indicated in FIG. 2 , i.e. based on thealignment of the C2c2 orthologues as indicated in FIG. 1 . As indicatedearlier, in certain embodiments, in the above amino acid residue list,the residues corresponding to R597, H602, R1278 and H1283 (referenced toLsh C2c2amino acids) are excluded.

The invention also provides for the one or more mutations or the two ormore mutations to be in a catalytically active domain of the effectorprotein. In certain embodiments, the one or more mutations or the two ormore mutations may be in a catalytically active domain of the effectorprotein comprising a HEPN domain, or a catalytically active domain whichis homologous to a HEPN domain. The effector protein may comprise one ormore heterologous functional domains. The one or more heterologousfunctional domains may comprise one or more nuclear localization signal(NLS) domains. The one or more heterologous functional domains maycomprise at least two or more NLS domains. The one or more NLS domain(s)may be positioned at or near or in proximity to a terminus of theeffector protein (e.g., C2c2) and if two or more NLSs, each of the twomay be positioned at or near or in proximity to a terminus of theeffector protein (e.g., C2c2). The one or more heterologous functionaldomains may comprise one or more translational activation domains. Inother embodiments the functional domain may comprise a transcriptionalactivation domain, for example VP64. The one or more heterologousfunctional domains may comprise one or more transcriptional repressiondomains. In certain embodiments the transcriptional repression domaincomprises a KRAB domain or a SID domain (e.g. SID4X). The one or moreheterologous functional domains may comprise one or more nucleasedomains. In a preferred embodiment a nuclease domain comprises Fok1.

The invention also provides for the one or more heterologous functionaldomains to have one or more of the following activities: methylaseactivity, demethylase activity, translation activation activity,translation repression activity, transcription activation activity,transcription repression activity, transcription release factoractivity, histone modification activity, nuclease activity,single-strand RNA cleavage activity, double-strand RNA cleavageactivity, single-strand DNA cleavage activity, double-strand DNAcleavage activity and nucleic acid binding activity. In certainembodiments of the invention, the one or more heterologous functionaldomains may comprise epitope tags or reporters. Non-limiting examples ofepitope tags include histidine (His) tags, V5 tags, FLAG tags, influenzahemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx)tags. Examples of reporters include, but are not limited to,glutathione-S-transferase (GST), horseradish peroxidase (HRP),chloramphenicol acetyltransferase (CAT) beta-galactosidase,beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed,DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP),and autofluorescent proteins including blue fluorescent protein (BFP).

At least one or more heterologous functional domains may be at or nearthe amino-terminus of the effector protein and/or wherein at least oneor more heterologous functional domains is at or near thecarboxy-terminus of the effector protein. The one or more heterologousfunctional domains may be fused to the effector protein. The one or moreheterologous functional domains may be tethered to the effector protein.The one or more heterologous functional domains may be linked to theeffector protein by a linker moiety.

The invention also provides for the effector protein comprising aneffector protein from an organism from a genus comprising Streptococcus,Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia,Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta,Lactobacillus, Eubacterium, Corynebacterium, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethylophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium orAcidaminococcus. The effector protein may comprise a chimeric effectorprotein comprising a first fragment from a first effector proteinortholog and a second fragment from a second effector protein ortholog,and wherein the first and second effector protein orthologs aredifferent. At least one of the first and second effector proteinorthologs may comprise an effector protein from an organism comprisingStreptococcus, Campylobacter, Nitratifractor, Staphylococcus,Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum,Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium,Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae,Clostridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus,Methanomethylophilus, Porphyromonas, Prevotella, Bacteroidetes,Helcococcus, Leptospira, Desulfovibrio, Desulfonatronum, Opitutaceae,Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium orAcidaminococcus.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may originate from,may be isolated from, or may be derived from a bacterial speciesbelonging to the taxa alpha-proteobacteria, Bacilli, Clostridia,Fusobacteria and Bacteroidetes. In certain embodiments, the effectorprotein, particularly a Type VI loci effector protein, more particularlya C2c2p, may originate from, may be isolated from, or may be derivedfrom a bacterial species belonging to a genus selected from the groupconsisting of Lachnospiraceae, Clostridium, Carnobacterium,Paludibacter, Listeria, Leptotrichia, and Rhodobacter. In certainembodiments, the effector protein, particularly a Type VI loci effectorprotein, more particularly a C2c2p may originate from, may be isolatedfrom or may be derived from a bacterial species selected from the groupconsisting of Lachnospiraceae bacterium MA2020, Lachnospiraceaebacterium NK4A179, Clostridium aminophilum (e.g., DSM 10710),Lachnospiraceae bacterium NK4A144, Carnobacterium gallinarum (e.g., DSM4847 strain MT44), Paludibacter propionicigenes (e.g., WB4), Listeriaseeligeri (e.g., serovar ½b str. SLCC3954), Listeria weihenstephanensis(e.g., FSL R9-0317 c4), Listeria newyorkensis (e.g., strain FSL M6-0635:also “LbFSL”), Leptotrichia wadei (e.g., F0279: also “Lw” or “Lw2”),Leptotrichia buccalis (e.g., DSM 1135), Leptotrichia sp. Oral taxon 225(e.g., str. F0581), Leptotrichia sp. Oral taxon 879 (e.g., strainF0557), Leptotrichia shahii (e.g., DSM 19757), Rhodobacter capsulatus(e.g., SB 1003, R121, or DE442). In certain preferred embodiments, theC2c2 effector protein originates from Listeriaceae bacterium (e.g. FSLM6-0635: also “LbFSL”), Lachnospiraceae bacterium MA2020,Lachnospiraceae bacterium NK4A179, Clostridium aminophilum (e.g., DSM10710), Carnobacterium gallinarum (e.g., DSM 4847), Paludibacterpropionicigenes (e.g., WB4), Listeria seeligeri (e.g., serovar ½b str.SLCC3954), Listeria weihenstephanensis (e.g., FSL R9-0317 c4),Leptotrichia wadei (e.g., F0279: also “Lw” or “Lw2”), Leptotrichiashahii (e.g., DSM 19757), Rhodobacter capsulatus (e.g., SB 1003, R121,or DE442); preferably Listeriaceae bacterium FSL M6-0635 (i.e. Listerianewyorkensis FSL M6-0635: “LbFSL” in FIGS. 4-7 ) or Leptotrichia wadeiF0279 (also “Lw” or “Lw2”).

In certain embodiments, a Type VI locus as intended herein may encodeCas1, Cas2, and the C2c2p effector protein.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, such as a nativeC2c2p, may be about 1000 to about 1500 amino acids long, such as about1100 to about 1400 amino acids long, e.g., about 1000 to about 1100,about 1100 to about 1200 amino acids long, or about 1200 to about 1300amino acids long, or about 1300 to about 1400 amino acids long, or about1400 to about 1500 amino acids long, e.g., about 1000, about 1100, about1200, about 1300, about 1400 or about 1500 amino acids long.

In certain embodiments, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, comprises at least oneand preferably at least two, such as more preferably exactly two,conserved RxxxxH motifs. Catalytic RxxxxH motifs are characteristic ofHEPN (Higher Eukaryotes and Prokaryotes Nucleotide-binding) domains.Hence, in certain embodiments, the effector protein, particularly a TypeVI loci effector protein, more particularly a C2c2p, comprises at leastone and preferably at least two, such as more preferably exactly two,HEPN domains. In certain embodiments, the HEPN domains may possess RNAseactivity. In other embodiments, the HEPN domains may possess DNAseactivity.

In certain embodiments, Type VI loci as intended herein may compriseCRISPR repeats between 30 and 40 bp long, more typically between 35 and39 bp long, e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp long.In particular embodiments, the direct repeat is at least 25 nt long.

In certain embodiments, a protospacer adjacent motif (PAM) or PAM-likemotif directs binding of the effector protein complex as disclosedherein to the target locus of interest. In some embodiments, the PAM maybe a 5′ PAM (i.e., located upstream of the 5′ end of the protospacer).In other embodiments, the PAM may be a 3′ PAM (i.e., located downstreamof the 5′ end of the protospacer). The term “PAM” may be usedinterchangeably with the term “PFS” or “protospacer flanking site” or“protospacer flanking sequence”.

In a preferred embodiment, the effector protein, particularly a Type VIloci effector protein, more particularly a C2c2p, may recognize a 3′PAM. In certain embodiments, the effector protein, particularly a TypeVI loci effector protein, more particularly a C2c2p, may recognize a 3′PAM which is 5′ H, wherein H is A, C or U. In certain embodiments, theeffector protein may be Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2, and the 5′ PAM is a 5′ H. In certainembodiments, the effector protein may be Leptotrichia wadei F0279 (Lw2)C2c2, and the 5′PAM is H, wherein H is C, U or A.

In certain embodiments, the CRISPR enzyme is engineered and can compriseone or more mutations that reduce or eliminate a nuclease activity.Mutations can also be made at neighboring residues, e.g., at amino acidsnear those indicated above that participate in the nuclease activity. Insome embodiments, only one HEPN domain is inactivated, and in otherembodiments, a second HEPN domain is inactivated.

In certain embodiments of the invention, the guide RNA or mature crRNAcomprises, consists essentially of, or consists of a direct repeatsequence and a guide sequence or spacer sequence. In certainembodiments, the guide RNA or mature crRNA comprises, consistsessentially of, or consists of a direct repeat sequence linked to aguide sequence or spacer sequence. In certain embodiments the guide RNAor mature crRNA comprises 19 nts of partial direct repeat followed by18, 19, 20, 21, 22, 23, 24, 25, or more nt of guide sequence, such as18-25, 19-25, 20-25, 21-25, 22-25, or 23-25 nt of guide sequence orspacer sequence. In certain embodiments, the effector protein is a C2c2effector protein and requires at least 16 nt of guide sequence toachieve detectable DNA cleavage and a minimum of 17 nt of guide sequenceto achieve efficient DNA cleavage in vitro. In particular embodiments,the effector protein is a C2c2 protein and requires at least 19 nt ofguide sequence to achieve detectable RNA cleavage. In certainembodiments, the direct repeat sequence is located upstream (i.e., 5′)from the guide sequence or spacer sequence. In a preferred embodimentthe seed sequence (i.e. the sequence essential critical for recognitionand/or hybridization to the sequence at the target locus) of the C2c2guide RNA is approximately within the first 5 nt on the 5′ end of theguide sequence or spacer sequence.

In preferred embodiments of the invention, the mature crRNA comprises astem loop or an optimized stem loop structure or an optimized secondarystructure. In preferred embodiments the mature crRNA comprises a stemloop or an optimized stem loop structure in the direct repeat sequence,wherein the stem loop or optimized stem loop structure is important forcleavage activity. In certain embodiments, the mature crRNA preferablycomprises a single stem loop. In certain embodiments, the direct repeatsequence preferably comprises a single stem loop. In certainembodiments, the cleavage activity of the effector protein complex ismodified by introducing mutations that affect the stem loop RNA duplexstructure. In preferred embodiments, mutations which maintain the RNAduplex of the stem loop may be introduced, whereby the cleavage activityof the effector protein complex is maintained. In other preferredembodiments, mutations which disrupt the RNA duplex structure of thestem loop may be introduced, whereby the cleavage activity of theeffector protein complex is completely abolished.

In particular embodiments, the C2c2 protein is an LshC2c2 effectorprotein and the mature crRNA comprises a stem loop or an optimized stemloop structure. In particular embodiments, the direct repeat of thecrRNA comprises at least 25 nucleotides comprising a stem loop. Inparticular embodiments, the the stem is amenable to individual baseswaps but activity is disrupted by most secondary structure changes ortruncation of the crRNA. Examples of disrupting mutations includeswapping of more than two of the stem nucleotides, addition of anon-pairing nucleotide in the stem, shortening of the stem (by removalof one of the pairing nucleotides) or extending the stem (by addition ofone set of pairing nucleotides). However, the crRNA may be amenable to5′ and/or 3′ extensions to include non-functional RNA sequences asenvisaged for particular applications described herein.

The invention also provides for the nucleotide sequence encoding theeffector protein being codon optimized for expression in a eukaryote oreukaryotic cell in any of the herein described methods or compositions.In an embodiment of the invention, the codon optimized nucleotidesequence encoding the effector protein encodes any C2c2 discussed hereinand is codon optimized for operability in a eukaryotic cell or organism,e.g., such cell or organism as elsewhere herein mentioned, for instance,without limitation, a yeast cell, or a mammalian cell or organism,including a mouse cell, a rat cell, and a human cell or non-humaneukaryote organism, e.g., plant.

In certain embodiments of the invention, at least one nuclearlocalization signal (NLS) is attached to the nucleic acid sequencesencoding the C2c2 effector proteins. In preferred embodiments at leastone or more C-terminal or N-terminal NLSs are attached (and hencenucleic acid molecule(s) coding for the C2c2 effector protein caninclude coding for NLS(s) so that the expressed product has the NLS(s)attached or connected). In certain embodiments of the invention, atleast one nuclear export signal (NES) is attached to the nucleic acidsequences encoding the C2c2 effector proteins. In preferred embodimentsat least one or more C-terminal or N-terminal NESs are attached (andhence nucleic acid molecule(s) coding for the C2c2 effector protein caninclude coding for NES(s) so that the expressed product has the NES(s)attached or connected). In a preferred embodiment a C-terminal and/orN-terminal NLS or NES is attached for optimal expression and nucleartargeting in eukaryotic cells, preferably human cells. In a preferredembodiment, the codon optimized effector protein is C2c2 and the spacerlength of the guide RNA is from 15 to 35 nt. In certain embodiments, thespacer length of the guide RNA is at least 16 nucleotides, such as atleast 17 nucleotides, preferably at least 18 nt, such as preferably atleast 19 nt, at least 20 nt, at least 21 nt, or at least 22 nt. Incertain embodiments, the spacer length is from 15 to 17 nt, from 17 to20 nt, from 20 to 24 nt, eg. 20, 21, 22, 23, or 24 nt, from 23 to 25 nt,e.g., 23, 24, or 25 nt, from 24 to 27 nt, from 27-30 nt, from 30-35 nt,or 35 nt or longer. In certain embodiments of the invention, the codonoptimized effector protein is C2c2 and the direct repeat length of theguide RNA is at least 16 nucleotides. In certain embodiments, the codonoptimized effector protein is C2c2 and the direct repeat length of theguide RNA is from 16 to 20 nt, e.g., 16, 17, 18, 19, or 20 nucleotides.In certain preferred embodiments, the direct repeat length of the guideRNA is 19 nucleotides.

The invention also encompasses methods for delivering multiple nucleicacid components, wherein each nucleic acid component is specific for adifferent target locus of interest thereby modifying multiple targetloci of interest. The nucleic acid component of the complex may compriseone or more protein-binding RNA aptamers. The one or more aptamers maybe capable of binding a bacteriophage coat protein. The bacteriophagecoat protein may be selected from the group comprising Qβ, F2, GA, fr,JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP,FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s and PRR1. Ina preferred embodiment the bacteriophage coat protein is MS2. Theinvention also provides for the nucleic acid component of the complexbeing 30 or more, 40 or more or 50 or more nucleotides in length.

Accordingly, it is an object of the invention not to encompass withinthe invention any previously known product, process of making theproduct, or method of using the product such that Applicants reserve theright and hereby disclose a disclaimer of any previously known product,process, or method. It is further noted that the invention does notintend to encompass within the scope of the invention any product,process, or making of the product or method of using the product, whichdoes not meet the written description and enablement requirements of theUSPTO (35 U.S.C. § 112, first paragraph) or the EPO (Article 83 of theEPC), such that Applicants reserve the right and hereby disclose adisclaimer of any previously described product, process of making theproduct, or method of using the product. It may be advantageous in thepractice of the invention to be in compliance with Art. 53(c) EPC andRule 28(b) and (c) EPC. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

In a further aspect, the invention provides a eukaryotic cell comprisinga nucleotide sequence encoding the CRISPR system described herein whichensures the generation of a modified target locus of interest, whereinthe target locus of interest is modified according to in any of theherein described methods. A further aspect provides a cell line of saidcell. Another aspect provides a multicellular organism comprising one ormore said cells.

In certain embodiments, the modification of the target locus of interestmay result in: the eukaryotic cell comprising altered (protein)expression of at least one gene product; the eukaryotic cell comprisingaltered (protein) expression of at least one gene product, wherein the(protein) expression of the at least one gene product is increased; theeukaryotic cell comprising altered (protein) expression of at least onegene product, wherein the (protein) expression of the at least one geneproduct is decreased; or the eukaryotic cell comprising an editedtranscriptome.

In certain embodiments, the eukaryotic cell may be a mammalian cell or ahuman cell.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA sequence-specificinterference, RNA sequence specific modulation of expression (includingisoform specific expression), stability, localization, functionality(e.g. ribosomal RNAs or miRNAs), etc.; or multiplexing of suchprocesses.

In further embodiments, the the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for RNA detection and/orquantification in a sample, such as a biological sample. In certainembodiments, RNA detection is in a cell. In an embodiment, the inventionprovides a method of detecting a target RNA in a sample, comprising (a)incubating the sample with i) a Type VI CRISPR-Cas effector proteincapable of cleaving RNA, ii) a guide RNA capable of hybridizing to thetarget RNA, and iii) an RNA-based cleavage inducible reporter capable ofbeing non-specifically and detectably cleaved by the effector protein,(b) detecting said target RNA based on the signal generated by cleavageof said RNA-based cleavage inducible reporter.

In an embodiment the Type VI CRISPR-Cas effector protein is a C2c2effector protein. In an embodiment, the RNA-based cleavage induciblereporter construct comprises a fluorochrome and a quencher. In certainembodiments, the sample comprises a cell-free biological sample. Inother embodiments, the sample comprises or a cellular sample, forexample, without limitation a plant cell, or an animal cell. In anembodiment of the invention, the target RNA comprises a pathogen RNA,including, but not limited to a target RNA from a virus, bacteria,fungus, or parasite. In an embodiment, the guide RNA is designed todetect a target RNA which comprises a single nucleotide polymorphism ora splice variant of an RNA transcript. In an embodiment, the guide RNAcomprises one or more mismatched nucleotides with the target RNA. Incertain embodiments, the guide RNA hybridizes to aa target molecule thatis diagnostic for a disease state, such as, but not limited to, cancer,or an immune disease.

The invention provides a ribonucleic acid (RNA) detection system,comprising a) a Type VI CRISPR-Cas effector protein capable of cleavingRNA, b) a guide RNA capable of binding to a target RNA, and c) anRNA-based cleavage inducible reporter capable of being non-specificallyand detectably cleaved by the effector protein. Further, the inventionprovides a kit for RNA detection, which comprises a) a Type VICRISPR-Cas effector protein capable of cleaving RNA, and b) an RNA-basedcleavage inducible reporter capable of being non-specifically anddetectably cleaved by the effector protein. In certain embodiments, theRNA-based cleavage inducible reporter construct comprises a fluorochromeand a quencher.

In further embodiments, the the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for generating disease modelsand/or screening systems.

In further embodiments, the non-naturally occurring or engineeredcompositions, the vector systems, or the delivery systems as describedin the present specification may be used for: site-specifictranscriptome editing or perturbation; nucleic acid sequence-specificinterference; or multiplexed genome engineering.

Also provided is a gene product from the cell, the cell line, or theorganism as described herein. In certain embodiments, the amount of geneproduct expressed may be greater than or less than the amount of geneproduct from a cell that does not have altered expression or editedgenome. In certain embodiments, the gene product may be altered incomparison with the gene product from a cell that does not have alteredexpression or edited genome.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1A-1D. Cellular localization of C2c2 orthologues. (A) HEK293 cellswere transfected with different C2c2 orthologues fused to mCherry withor without nuclear localization signal (NLS) or nuclear export signal(NES). (B) NES fusion of Leptotrichia wadei F0279 C2c2 (B1) andLachnospiraceae bacterium NK4A179 C2c2 (B2) locates in the cytoplasm.(C) NLS fusion of Leptotrichia wadei F0279 C2c2 (C1), Lachnospiraceaebacterium NK4A179 C2c2 (C2), and Leptotrichia shahii C2c2 (C3) locatesin the nucleus and variably in the nucleolus. (C) Leptotrichia wadeiF0279 C2c2 (D1), Lachnospiraceae bacterium NK4A179 C2c2 (D2), andLeptotrichia shahii C2c2 (D3) without fusion to NLS or NES variablylocates in the nucleus and cytoplasm. (D) Schematic of the fourmammalian LwaCas13a constructs evaluated (left) and imaging showing thelocalization and expression of each of the designs (right).

FIG. 2A-2C. Assay for evaluation of C2c2 knockdown efficiency inmammalian cells. (A) Schematic of the dual luciferase reporter schemeused to determine knockdown efficiency of C2c2. Cells were transfectedwith different plasmids: a plasmid encoding Gaussia luciferase (Gluc)and Cypridina luciferase (Cluc); a plasmid encoding C2c2 (with orwithout NLS or NES); and a plasmid encoding a gRNA targeting Gaussialuciferase. Knockdown efficiency of Gluc is determined based on theratio of Gluc and Cluc. (B) positions of different gRNAs targetingGaussia luciferase mRNA. (C) Assay for evaluation of C2c2 knockdownefficiency in mammalian cells. Cells were transfected with differentplasmids: a plasmid encoding EGFP; a plasmid encoding C2c2 (with orwithout NLS or NES); and a plasmid encoding a gRNA targeting EGFP.Knockdown efficiency of EGFP is determined by flow cytometry.

FIG. 3A-3Q. Characterization of the type VI CRISPR Cas13a family for RNAcleavage activity. (A) Schematic of PFS characterization screen onCas13a orthologs. (B) Schematic of constructs for expression of Cas13aprotein and crRNA. Length and residue numbers shown are for LwaCas13a.(C) Quantitation of Cas13a in vivo activity. (D) Ratios of in vivoactivity from (C). (E, F) The PFS motif determination for LshC2c2 andLwaC2c2, as determined by next generation sequencing of enrichedplasmids in the surviving E. coli population. (G) Distributions of PFSenrichment for LshCas13a and LwaCas13a in targeting and non-targetingsamples. (H) In vivo PFS screening shows LwaCas13a has a minimal PFSpreference. (I) Box plot showing the distribution of normalized PFScounts for targeting and non-targeting bio-replicates (n=2) for LshC2c2and LwC2c2. The box extends from the first to third quartile withwhiskers denoting 1.5 times the interquartile range. The mean isindicated by the red horizontal bar. (J) Comparison of LshC2c2 andLwC2c2 RNA cleavage activity using purified protein. LwaCas13a has moreactive RNAse activity than LshCas13a. A 5′ labeled RNA target wasincubated with C2c2 and corresponding crRNA for 30 min and the productswere analyzed by gel electrophoresis. (K) LwaCas13a can process CRISPRarray transcripts from the L. wadei CRISPR locus. A two-spacer Lwa arraywas incubated with LwC2c2 for 30 min and the reaction was then resolvedby gel electrophoresis. (L) Schematic of mammalian LwaCas13a constructsevaluated and imaging showing the localization and expression of each ofthe designs. Scale bars, 10 μm. (M) Schematic of the mammalian reportersystem used to evaluate knockdown by a luciferase protein readout. (N)Knockdown of Gaussia luciferase (Gluc) using engineered variants ofLwaCas13a. Sequences for guides (SEQ ID NOS: 410 and 411, respectively,in order of appearance) and shRNAs are shown above. (O) Knockdown ofthree different endogenous transcripts with LwaCas13a compared againstcorresponding RNAi constructs. (P) Schematic for LwaCas13a knockdown oftranscripts in rice (Oryza sativa) protoplasts. (Q) LwaCas13a knockdownof three transcripts in Oryza sativa protoplasts using three targetingguides and a non-targeting guide per transcript. All values are mean±SEMwith n=3, unless otherwise noted.

FIG. 4A-4B. Normalized protein expression of luciferase with differentgRNAs directed against Gluc and with C2c2 orthologues fused with NLS,NES, or no tag. (A) C2c2 orthologue is Leptotrichia wadei F0279 (Lw2).Spacer sequences used in the experiments are (Guide 1)ATCAGGGCAAACAGAACTTTGACTCCCA (SEQ ID NO: 1); (Guide 2)AGATCCGTGGTCGCGAAGTTGCTGGCCA (SEQ ID NO: 2); (Guide 3)TCGCCTTCGTAGGTGTGGCAGCGTCCTG (SEQ ID NO: 3); and (Guide NP)TAGATTGCTGTTCTACCAAGTAATCCAT (SEQ ID NO: 4). (B) C2c2 orthologue isListeria newyorkensis FSL M6-0635 (LbFSL). Spacer sequences used in theexperiments are (Guide 1) TCGCCTTCGTAGGTGTGGCAGCGTCCTG (SEQ ID NO: 5)and (Guide NT) tagattgctgttctaccaagtaatccat (SEQ ID NO: 6).

FIG. 5A-5B. Normalized protein expression of GFP with different gRNAsdirected against EGFP and with C2c2 orthologues fused with NLS, NES, orno tag. (A) C2c2 orthologues is Leptotrichia wadei F0279 (Lw2). Spacersequences used in the experiments are (Guide 1)tgaacagctcctcgcccttgctcaccat (SEQ ID NO: 7); (Guide 2)tcagcttgccgtaggtggcatcgccctc (SEQ ID NO: 8); (Guide 3)gggtagcggctgaagcactgcacgccgt (SEQ ID NO; 9); (Guide 4)ggtcttgtagttgccgtcgtccttgaag (SEQ ID NO: 10); (Guide 5)tactccagcttgtgccccaggatgttgc (SEQ ID NO: 11); (Guide 6)cacgctgccgtcctcgatgttgtggcgg (SEQ ID NO: 12); (Guide 7)tctttgctcagggcggactgggtgctca (SEQ ID NO: 13); (Guide 8)gacttgtacagctcgtccatgccgagag (SEQ ID NO: 14); and (Guide NT)tagattgctgttctaccaagtaatccat (SEQ ID NO: 6). (B) C2c2 orthologues isListeria newyorkensis FSL M6-0635 (LbFSL). Spacer sequences used in theexperiments are (Guide 2) tcagcttgccgtaggtggcatcgccctc (SEQ ID NO: 8);(Guide 3) gggtagcggctgaagcactgcacgccgt (SEQ ID NO: 9); (Guide 4)ggtcttgtagttgccgtcgtccttgaag (SEQ ID NO: 10); (Guide 5)tactccagcttgtgccccaggatgttgc (SEQ ID NO: 11); (Guide 6)cacgctgccgtcctcgatgttgtggcgg (SEQ ID NO: 12); (Guide 7)tctttgctcagggcggactgggtgctca (SEQ ID NO: 13); (Guide 8)gacttgtacagctcgtccatgccgagag (SEQ ID NO: 14); and (Guide NT)tagattgctgttctaccaagtaatccat (SEQ ID NO: 6).

FIG. 6A-6E. Engineering and optimization of LwaCas13a for mammalianknockdown. (A) Knockdown of Gluc transcript by LwCas13a using a varietyof guides transfected in A375s cells.: (B) Knockdown of Gaussialuciferase (Gluc) using engineered variants of LwaCas13a. (C) Knockdownof Gaussia luciferase (Gluc) by LwaCas13a and Gluc crRNA 1 spacers ofvarying lengths. (D) Knockdown of KRAS transcript by LwaCas13a using avariety of guides. (E) Knockdown of PPIB transcript by LwaCas13a using avariety of guides.

FIG. 7 . Normalized protein expression of target genes with gRNAsdirected against the respective target genes and with C2c2 Leptotrichiawadei F0279 fused with NES. gRNAs for respective target genes are:ctgctgccacagaccgagaggcttaaaa (CTNNB1) (SEQ ID NO: 15);tccttgattacacgatggaatttgctgt (PPIB) (SEQ ID NO: 16);tcaaggtggggtcacaggagaagccaaa (mAPK14) (SEQ ID NO: 17);atgataatgcaatagcaggacaggatga (CXCR4) (SEQ ID NO; 18);gcgtgagccaccgcgcctggccggctgt (TINCR) (SEQ ID NO: 19);ccagctgcagatgctgcagtttttggcg (PCAT1) (SEQ ID NO: 20);ctggaaatggaagatgccggcatagcca (CAPN1) (SEQ ID NO: 21);gatgacacctcacacggaccacccctag (LETMDI) (SEQ ID NO: 22);taatactgctccagatatgggtgggcca (MAPK14) (SEQ ID NO: 23);catgaagaccgagttatagaatactata (RB1) (SEQ ID NO: 24);ggtgaaatattctccatccagtggtttc (TP53) (SEQ ID NO: 25); andaatttctcgaactaatgtatagaaggca (KRAS) (SEQ ID NO: 26).

FIG. 8 . Normalized protein expression of luciferase with differentgRNAs directed against Gluc and with C2c2 orthologues fused with NLS,NES, or no tag. C2c2 orthologues are Leptotrichia shahii (Lsh),Leptotrichia wadei F0279 (Lw2), Clostridium aminophilum (Ca), Listerianewyorkensis FSL M6-0635 (LbFSL), Lachnospiraceae bacterium NK4A179(LbNk), and Lachnospiraceae bacterium MA2020 (LbM).

FIG. 9 . Lw2 functions in multiple cell lines. Normalized luciferaseactivity is shown for 293FT cells.

FIG. 10A-10B. Relative protein expression of luciferase with gRNAdirected against Gluc and with catalytically inactivated C2c2orthologues. (A) dC2c2 Leptotrichia wadei (LwC2c2) was fused to a EIF4E,EIF4E and NES, or no tag. (B) dC2c2 Listeria newyorkensis FSL M6-0635(LbFSL) was fused to a NLS and EIF4E or no tag.

FIG. 11A-11C. Cellular localization of Leptotrichia wadei C2c2 targetingbeta actin localizes to stress granules upon treatment of cells withNaAsO₂.

FIG. 12 . RNA knockdown in mammalian cells. C2c2 outperforms shRNA attwo target sites.

FIG. 13 . Increasing crRNA transfection amount increases knockdown.

FIG. 14 . Lw2C2c2 with NES (NES-Lw2C2c2) effectively cleaves RNA of tRNAand U6 knockdown.

FIG. 15A-15B. (A) Protein transfection amount saturates knockdown. (B)Knockdown of Gluc transcript with Gluc crRNA 1 and varying amounts oftransfected LwaCas13a plasmid.

FIG. 16 . U6-driven DR-spacer-DR-spacer targeting constructs.

FIG. 17 . Optimized shRNA is outperformed by C2c2 for correspondingtargets on endogenous genes.

FIG. 18 . dLw2C2c2-EIF4E fusion can upregulate translation of threegenes; Protein levels as measured by band intensity on western blot.

FIG. 19 . Knock-down of individual transcripts. Lw2 shows lessvariability and higher specificity.

FIG. 20 . RNA knockdown in mammalian cells.

FIG. 21 . Imaging is improved by a superfolding derivative of GFP(sfGFP). C2c2-mCherry and sfGFP-C2c2 fusion proteins are compared inHEK293FT cells and mouse embryonic stem cells (mESC).

FIG. 22A. msfGFP-C2c2 improves knockdown. Luciferase knockdown byvarious fusion proteins of C2c2 with mCherry or msfGFP, furtherincluding an NLS or NES, are depicted.

FIG. 22B-22D illustrates a protein tagging system for regulatingtranscription with C2c2 and shows elements of a transcription initiationfactor-linked scFv binding to short peptide sequences comprised by amodified C2c2 (SunTag). FIG. 22C-22D depicts transcriptional effects ofthe system.

FIG. 23A-23B. Splitting of reporter proteins (GFP, Venus, Cre etc.).Each part is fused to C2c2 (see slide for example schematic withCaspase). By designing two guides that target a transcript close to eachother, the split protein is reconstituted in the presence of thetranscript.

FIG. 24A-24C. Rapid RNA detection by C2c2 collateral RNase activity. (A)Left panel: Activation of C2c2 collateral non-specific RNase activity bytarget RNA complementary to guide sequence of C2c2 crRNA leads tocleavage of reporter. Right panel: In the absence of target RNA,collateral non-specific RNase activity is not induced, hence there is nocleavage of reporter. (B) Schematic of assay for detecting the activityof the collateral effect by LwaCas13a. (C) Gel electrophoresis ofcollateral RNA targets after incubation with LwaCas13a-crRNA complex inthe presence or absence of target RNA.

FIG. 25 . Lentivirus detection by collateral effect using variousconcentrations of C2c2.

FIG. 26 . Time course of lentivirus detection by collateral effect.

FIG. 27 . Detection of rare RNA species by C2c2 collateral RNaseactivity. Increasing target concentration and target cleavage isaccompanied by increased non-specific off-target RNase activity.

FIG. 28A-28B. Alignment of Lw2C2c2 (SEQ ID NO: 412) and LbuC2c2 (SEQ IDNO: 74).

FIG. 29A-29C. (A) PLwC2c2 targeted for luciferase mRNA knockdown withsingle base-pair mismatches in the spacer sequence. (B) LwC2c2 targetedfor CXCR4 mRNA knockdown with single base-pair mismatches in the spacersequence. (C) LwC2c2 targeted for luciferase mRNA knockdown with singleand consecutive double base-pair mismatches in the spacer sequence.

FIG. 29D-I to 29D-III. (I) Specificity of knockdown of luciferasereporter mRNA. C2c2 is more specific than RNAi for knockdown ofluciferase reporter mRNA. Left: Expression levels in log 2(transcriptsper million (TPM)) values of all detected genes in RNA-seq libraries ofnon-targeting-transfected controls (x axis of all graphs) compared tothe luciferase knockdown condition for shRNA. Right: Expression levelsin log 2(transcripts per million (TPM)) values of all detected genes inRNA-seq libraries of non-targeting-transfected controls (x axis of allgraphs) compared to the luciferase knockdown condition for C2c2. Thetarget luciferase transcript targeted for knockdown is indicated by thered dot. The average from n=3 biological replicates is shown. (II)Specificity of knockdown of endogenous KRAS mRNA. C2c2 is more specificthan RNAi for knockdown of endogenous KRAS. The target transcript isindicated by the red dot. (III) Specificity of knockdown of endogenousPPIB mRNA. C2c2 is more specific than RNAi for knockdown of endogenousPPIB. The target transcript is indicated by the red dot.

FIG. 29E-29G. (E) Differential gene expression analysis of six RNA-seqlibraries (each with three biological replicates) comparing LwaCas13aknockdown to shRNA knockdown at three different genes. Genes areconsidered significantly differentially expressed if they have a meanfold change >2 or <0.75 compared to non-targeting controls and have afalse-discovery rate (FDR) <0.10. (F) Quantified mean knockdown levelsfor the targeted genes from the RNA seq libraries. (G) Left: Luciferaseknockdown for cells transfected with LwaCas13a for 72 hours with andwithout antibiotic selection prior to measuring cell growth. Middle:Cell viability for cells that have been transfected with LwaCas13a for72 hours with and without antibiotic selection. Right: GFP fluorescenceof cells that have been transfected with LwaCas13a for 72 hours with andwithout antibiotic selection. All values are mean±SEM with n=3.

FIG. 30A-30E. Comparison of level and specificity of RNA knockdown. C2c2demonstrates increased specificity while maintaining similar levels ofknockdown as RNAi. (A) The number of significant differentiallyregulated genes in each RNA-seq library analyzed. Differential geneexpression analysis of six RNA-seq libraries (each with three biologicalreplicates) comparing LwaCas13a knockdown to shRNA knockdown at threedifferent genes. Genes are considered significantly differentiallyexpressed if they have a mean fold change >2 or <0.75 compared tonon-targeting controls and have a false-discovery rate (FDR)<0.10. (B)Quantified mean knockdown levels for the targeted genes from the RNA seqlibraries. Normalized expression is calculated as the TPM of thetargeted gene in the targeting condition divided by the TPM of thetargeted gene in the non-targeted condition. The plots representaccumulated data of RNA-seq plots in FIG. 28 . Off-targets aredetermined as genes that are significantly unregulated (>2 fold) ordown-regulated (<6969 (C) Luciferase knockdown for cells transfectedwith LwaCas13a for 72 hours with a non-selectable andblasticidin-selectable version of C2c2. (D) Cell viability for cellsthat have been transfected with LwaCas13a for 72 hours with and withoutantibiotic selection. (E) GFP fluorescence of cells that have beentransfected with LwaCas13a for 72 hours with and without antibioticselection.

FIG. 3I. C2c2 is capable of multiplexed knockdown. crRNA was designed totarget PPIB, CXCR4, KRAS, TINCR, and PCAT. The top panel showsnormalized expression levels of the mRNAs color-coded and in the sameorder left-to-right as the depicted multiplexed crRNA transcript. Thebottom panel compares singly dosed guides with multiplexed and pooledguides.

FIG. 32A-32D. Tiling guides along the length of gLuc or cLucdemonstrates retargetability and regions of vulnerability on transcript.(A) Schematic of LwaCas13a arrayed screening (SEQ ID NOS: 413-418,respectively, in order of appearance). (B) Knockdown efficiency ofGaussia luciferase mRNA by LwC2c2 with 186 guides tiled evenly acrossthe length of the transcript. (C) Arrayed knockdown screen of Cypridinaluciferase by LwC2c2 with guides tiled evenly across the length of thetranscript. (D) Validation of the top three crRNAs from the arrayedknockdown screens with shRNA comparisons.

FIG. 33A-33B. RNA Immunoprecipitation (RIP) shows enrichment of targetbinding. dC2c2-msfGFP comprising an HA-tag provided for precipitation ofthe protein-RNA complex. Bound RNA was quantified by qPCR and enrichmentdetermined in comparison to input RNA (sample withoutimmunoprecipitation) and immunoprecipitation with a negative controlantibody. Panel A shows enrichment of beta-actin RNA target. Panel Bshows enrichment of luciferase target.

FIG. 34 . C2c2 imaging of Beta-actin in 3T3L1 fibroblasts. In thetargeting condition (top frames) C2c2 complex binds actin mRNA leavingthe nucleus, revealing cytoplasmic Beta-actin mRNA. In the non-targetingcondition, NLS-tagged C2c2 is observed in the nucleus. Left and rightcolumns constitute different views.

FIG. 35 . C2c2 imaging of Beta-actin in HEK293FT cells. In the targetingcondition (top frames) C2c2 complex binds actin mRNA leaving thenucleus, revealing cytoplasmic Beta-actin mRNA. In the non-targetingcondition, NLS-tagged C2c2 is observed in the nucleus. Left and rightcolumns constitute different views.

FIG. 36 . In vitro characterization of the RNA cleavage kinetics ofLw2Cas13a. A denaturing gel after 0.5 hour of RNA-cleavage of5′end-labeled target 1 using LwaCas13a-crRNA complex that is seriallydiluted in half-log steps.

FIG. 37 . In vitro characterization of the RNA cleavage kinetics ofLw2Cas13a. Denaturing gel of a time series of Lw2Cas13a ssRNA cleavageusing a 5′ end-labeled target 1.

FIG. 38 . Lw2C2c2 and crRNA mediate RNA-guided ssRNA cleavage. Adenaturing gel demonstrating crRNA-mediated ssRNA cleavage by Lw2 C2c2after 1 hour of incubation. The ssRNA target is 5′ labeled with IRDye800. Cleavage requires the presence of the crRNA and is abolished byaddition of EDTA.

FIG. 39A-39B. Lw2C2c2 and crRNA mediate RNA-guided ssRNA cleavage. (A)Schematic of the ssRNA substrate (SEQ ID NO: 419) being targeted by thecrRNA (SEQ ID NO: 420). The protospacer region is highlighted in blueand the PFS is indicated by the magenta bar. (B) A denaturing geldemonstrating the requirement for an PFS after 3 hours of incubation.Four ssRNA substrates that are identical except for the PFS (indicatedby the magenta X in the schematic) were used for the in vitro cleavagereactions. ssRNA cleavage activity is dependent on the nucleotideimmediately 3′ of the target site.

FIG. 40 . Direct repeat length affects the RNA-guided RNase activity ofLw2C2c2. Denaturing gel showing crRNA-guided cleavage of ssRNA 1 as afunction of direct repeat length after 3 hours of incubation. Figurediscloses SEQ ID NO: 421.

FIG. 41 . Spacer length affects the RNA-guided RNase activity ofLw2C2c2. Denaturing gel showing crRNA-guided cleavage of ssRNA 1 as afunction of spacer length after 3 hours of incubation. Figure disclosesSEQ ID NOS: 421-424, respectively, in order of appearance.

FIG. 42A-42C. Lw2C2c2 cleavage sites are determined by secondarystructure and sequence of the target RNA. (A) Denaturing gel showingssRNA 4 and ssRNA 5 after incubation with LwCas13a and crRNA 1. (B)ssRNA 4 (blue) (SEQ ID NO: 339) and (C) ssRNA 5 (SEQ ID NO: 340);(green) share the same protospacer but are flanked by differentsequences. Despite identical protospacers, different flanking sequencesresulted in different cleavage patterns.

FIG. 43A-43C. (A) Schematic of ssRNA 4 modified with a homopolymerstretch in the highlighted loop (red) for each of the four possiblenucleotides (SEQ ID NOS: 341-344). crRNA spacer sequence is highlightedin blue. (B). Denaturing gel showing Lw2C2c2-crRNA-mediated cleavage foreach of the four possible homopolymer targets after 3 hours ofincubation. Lw2C2c2 cleaves C and U. (C) LwaCas13a can process pre-crRNAfrom the L. wadei CRISPR-Cas locus.

FIG. 44A-44K. A catalytically-inactive LwaCas13a (dCas13a) is capable ofbinding transcripts in mammalian cells. (A) Schematic of dCas13a-GFPconstruct used for imaging and evaluation of dCas13a binding. (B)Schematic of RNA immunoprecipitation for quantitation of dCas13abinding. (C) dCas13a targeting gLuc transcripts is significantlyenriched compared to non-targeting controls. Quantification ofdC2c2-msfGFP binding for g Gaussia luciferase mRNA by RIP normalized toeither control antibody or input lysate. Values are normalized tonon-targeting guide. (n=3, *, p<0.05; ****, p<0.0001 by t-test). (D)Schematic of dCas13a-GFP-KRAB construct used for negative-feedbackimaging. In the absence of target and guide, the reporter proteininhibits its own transcription. (E) Gluc, Cluc, PPIB, and KRAS knockdownpartially correlates with target accessibility as measured by predictedfolding of the transcript. (F) Comparison between localization ofdCas13-GFP and dCas13a-GFP-KRAB constructs for imaging b-actin. (G)Representative images for dCas13a-GFP-KRAB imaging with multiple guidestargeting b-actin in HEK293 cells. (H) Representative images fordCas13a-GFP-KRAB imaging with multiple guides targeting ACTB. (I)Quantification of cytoplasmic translocation of dCas13a-GFP-KRAB, asmeasured by the ratio of nuclear to whole-cell signal. (J)Representative fixed immunofluorescence images of 293FT cells treatedwith 400 μM sodium arsenite. Stress granules are indicated by stainingfor marker G3BP1. Scale bars, 5 μm. Scale bars, 5 μm. (K) G3BP1 anddCas13a-GFP-KRAB co-localization quantified per cell by Pearson'scorrelation. All values are mean±SEM with n=3. ****p<0.0001; ***p<0.001;**p<0.01; *p<0.05. ns=not significant. A one-tailed student's t-test wasused for comparisons in (a) and a two-tailed student's t-test was usedfor comparisons in (I) and (K).

FIG. 45 . Detection of B-actin using a C2c2-GFP imaging protein as shownin FIG. 43 . β-actin was imaged using dC2c2-eGFP-ZF-KRAB-NLS with atargeting guide (left panel) of a non-targeting guide (right panel).

FIG. 46 . Detection of stress granules using GFP-tagged G3BP1.

FIG. 47 . Detection of acting in stress granules and stress granulesubstructures (“cores”). Actin mRNA was imaged using C2c2-eGFP-ZF-KRAB,with and without a targeting construct and with or without treatmentwith NaAsO₂ to stabilize core substructures. Stress granules werelabeled using anti-G3BP1.

FIG. 48A-48F: dCas13a can image stress granule formation in livingcells. (A) Schematic for using the negative-feedback dCas13a-msfGFP-KRABconstruct for imaging the localization of β-actin mRNA to stressgranules upon treatment with sodium arsenite. (B) Representative fixedimmunofluorescence images of 293FT cells treated with 400 μM sodiumarsenite. dCas13a-msfGFP-KRAB transfected along with β-actin mRNAtargeting guides localizes to stress granules. Shown are representativeimages of fixed HEK293 cells immunostained with antibodies against theG3BP1 marker for stress granules. (C) Quantification of stress granulelocalization by Pearson's correlation analysis of ˜20 cells percondition. (D) Quantification of stress granule localization by Manders'colocalization analysis of ˜20 cells per condition. (E) Representativeimages from live-cell analysis of stress granule formation in responseto 400 uM sodium arsenite treatment. (F) Quantitation of stress granuleformation in response to sodium arsenite treatment.

FIG. 49A-49O LwaCas13a can be reprogrammed to target endogenousmammalian coding and non-coding RNA targets. (A) Arrayed knockdownscreen of 93 guides evenly tiled across the KRAS transcript. (B) Arrayedknockdown screen of 93 guides evenly tiled across the PPIB transcript.(C) Schematic of LwaCas13a arrayed screening (SEQ ID NOS: 413, 414, 416and 418, respectively, in order of appearance). (D) Arrayed knockdownscreen of 186 guides evenly tiled across the Gluc transcript. (E)Arrayed knockdown screen of 93 guides evenly tiled across the Cypridinaluciferase (Cluc) transcript. (F) Arrayed knockdown screen of 93 guidesevenly tiled across the KRAS transcript. (G) Arrayed knockdown screen of93 guides evenly tiled across the PPIB transcript. (H) Validation of thetop three guides from the endogenous arrayed knockdown screens withshRNA comparisons. All values are mean±SEM with n=3. ***p<0.001;**p<0.01. A two-tailed student's T-test was used for comparisons. (I)Arrayed knockdown screen of 93 guides evenly tiled across the MALAT1transcript. (J) Validation of top three guides from the endogenousarrayed MALAT1 knockdown screen with shRNA comparisons. (K) Multiplexeddelivery of five guides in a CRISPR array against five differentendogenous genes under the expression of a single promoter is capable ofrobust knockdown. (L) Multiplexed delivery of three guides against threedifferent endogenous genes or with constructs replacing each of theguides with a non-targeting sequence shows specific knockdown of thegenes targeted. All values are mean±SEM with n=3. (M) Knockdown of threedifferent endogenous transcripts with LwaCas13a compared againstcorresponding RNAi constructs. (N) LwaCas13a is capable of knocking downthe nuclear lncRNA transcript MALAT1. (0) Multiplexed delivery of threeguides against three different endogenous genes or with constructsreplacing each of the crRNAs with a non-targeting sequence showsspecific knockdown of the genes targeted.

FIG. 50 . HEPN sequence motifs from 21 C2c2 orthologs.

FIG. 51 . HEPN sequence motifs from 33 C2c2 orthologs.

FIG. 52 . Exemplary locations for linkage of effector domains. C2c2proteins linked at the C or N terminus to heterologous functionaldomains (mCherry and msfGFP depicted) retain function as indicated byluciferase knockdown.

FIG. 53A-53L. (A-K) Sequence alignment of C2c2 orthologs (SEQ ID NOS:68, 69, 59-61, 80, 62-64, 73, 65, 66, 79, 71, 425 and 67, respectively,in order of appearance). (L) Sequence alignment of HEPN domains (SEQ IDNOS: 547-576, respectively, in order of appearance).

FIG. 54 . Tree alignment of C2c2 orthologs.

FIG. 55A-55B. Tree alignment of C2c2 and Cas13b orthologs.

FIG. 56A-56F: Engineering and optimization of LwaCas13a for mammalianknockdown. (A) Knockdown of Gluc transcript with Gluc crRNA 1 andvarying amounts of transfected LwaCas13a plasmid. (B) Knockdown of Gluctranscript by LwaCas13a and varying amounts of transfected Gluc crRNA 1and 2 plasmid. (C) Knockdown of Gluc transcript using crRNAs expressedfrom either U6 or tRNAVal promoters. (D) Knockdown of KRAS transcriptusing crRNAs expressed from either U6 or tRNAVal promoters. (E)Knockdown of KRAS transcript using guides expressed from either U6 ortRNA^(Val) promoters. (F) Arrayed knockdown screen of 93 guides evenlytiled across the XIST transcript.

FIG. 57A-57E: Evaluation of LwaCas13a PFS preferences and comparisons toLshCas13a. (A) Sequence comparison tree of the fifteen Cas13a orthologsevaluated in this study. (B) Number of LshCas13a and LwaCas13a PFSsequences above depletion threshold for varying depletion thresholds.(C) Distributions of PFS enrichment for LshCas13a and LwaCas13a intargeting samples, normalized to non-targeting samples. (D) Sequencelogos and counts for remaining PFS sequences after LshCas13a cleavage atvarying enrichment cutoff thresholds. (E) Sequence logos and counts forremaining PFS sequences after LwaCas13a cleavage at varying enrichmentcutoff thresholds.

FIG. 58A-58D: LwaCas13a targeting efficiency is influenced byaccessibility along the transcript. (A) First row: Top knockdown guidesare plotted by position along target transcript. The top 20% of guidesare chosen for Gluc and top 30% of guides for Cluc, KRAS, and PPIB.Second row: Histograms for the pairwise distance between adjacent topguides for each transcript (blue) compared to a random null-distribution(red). Inset shows the cumulative frequency curves for these histograms.A shift of the blue curve (actual measured distances) to the left of thered curve (null distribution of distances) indicates that guides arecloser together than expected by chance. (B) Gluc, Cluc, PPIB, and KRASknockdown partially correlates with target accessibility as measured bypredicted folding of the transcript. (C) Kernel density estimation plotsdepicting the correlation between target accessibility (probability of aregion being base-paired) and target expression after knockdown byLwaCas13a. (D) First row: Correlations between target expression andtarget accessibility (probability of a region being base-paired)measured at different window sizes (W) and for different k-mer lengths.Second row: P-values for the correlations between target expression andtarget accessibility (probability of a region being base-paired)measured at different window sizes (W) and for different k-mer lengths.The color scale is designed such that p-values >0.05 are shades of redand p-values <0.05 are shades of blue.

FIG. 59A-59K: Detailed evaluation of LwaCas13a sensitivity to mismatchesin the crRNA:target duplex at varying spacer lengths. (A) Knockdown ofKRAS evaluated with crRNAs containing single mismatches at varyingpositions across the spacer sequence. (B) Knockdown of PPIB evaluatedwith crRNAs containing single mismatches at varying positions across thespacer sequence. (C) Knockdown of Gluc evaluated with guides containingnon-consecutive double mismatches at varying positions across the spacersequence. The wild-type sequence is shown at the top with mismatchidentities shown below. Figure discloses SEQ ID NOS: 426-450,respectively, in order of appearance. (D) Collateral cleavage activityon ssRNA 1 and 2 for varying spacer lengths. (n=4 technical replicates;bars represent mean±s.e.m.). (E) Specificity ratios of crRNA tested in(D). Specificity ratios are calculated as the ratio of the on-target RNA(ssRNA 1) collateral cleavage to the off-target RNA (ssRNA 2) collateralcleavage. (n=4 technical replicates; bars represent mean±s.e.m.). (F)Collateral cleavage activity on ssRNA 1 and 2 for 28 nt spacer crRNAwith synthetic mismatches tiled along the spacer. (n=4 technicalreplicates; bars represent mean±s.e.m.). (G) Specificity ratios of crRNAtested in (F). Specificity ratios are calculated as the ratio of theon-target RNA (ssRNA 1) collateral cleavage to the off-target RNA (ssRNA2) collateral cleavage. (n=4 technical replicates; bars representmean±s.e.m.). (H) Collateral cleavage activity on ssRNA 1 and 2 for 23nt spacer crRNA with synthetic mismatches tiled along the spacer. (n=4technical replicates; bars represent mean±s.e.m.). (I) Specificityratios of crRNA tested in (H). Specificity ratios are calculated as theratio of the on-target RNA (ssRNA 1) collateral cleavage to theoff-target RNA (ssRNA 2) collateral cleavage. (n=4 technical replicates;bars represent mean±s.e.m.). (J) Collateral cleavage activity on ssRNA 1and 2 for 20 nt spacer crRNA with synthetic mismatches tiled along thespacer. (n=4 technical replicates; bars represent mean±s.e.m.). (K)Specificity ratios of crRNA tested in (J). Specificity ratios arecalculated as the ratio of the on-target RNA (ssRNA 1) collateralcleavage to the off-target RNA (ssRNA 2) collateral cleavage. (n=4technical replicates; bars represent mean±s.e.m.)

FIG. 60A-60F: LwaCas13a is more specific than shRNA knockdown onendogenous targets and has little variation comparable to biologicalnoise. (A) Left: Expression levels in log 2(transcripts per million(TPM)) values of all genes detected in RNA-seq libraries ofnon-targeting shRNA-transfected control (x-axis) compared toKRAS-targeting shRNA (y-axis). Shown is the mean of three biologicalreplicates. The KRAS transcript data point is colored in red. Right:Expression levels in log 2(transcripts per million (TPM)) values of allgenes detected in RNA-seq libraries of non-targetingLwaCas13a-crRNA-transfected control (x-axis) compared to KRAS-targetingLwaCas13a-crRNA (y-axis). Shown is the mean of three biologicalreplicates. The KRAS transcript data point is colored in red. (B) Left:Expression levels in log 2(transcripts per million (TPM)) values of allgenes detected in RNA-seq libraries of non-targeting shRNA-transfectedcontrol (x-axis) compared to PPIB-targeting shRNA (y-axis). Shown is themean of three biological replicates. The PPIB transcript data point iscolored in red. Right: Expression levels in log 2(transcripts permillion (TPM)) values of all genes detected in RNA-seq libraries ofnon-targeting LwaCas13a-crRNA-transfected control (x-axis) compared toPPIB-targeting LwaCas13a-crRNA (y-axis). Shown is the mean of threebiological replicates. The PPIB transcript data point is colored in red.(C) Comparisons of individual replicates of non-targeting shRNAconditions (first row) and Gluc-targeting shRNA conditions (second row).(D) Comparisons of individual replicates of non-targeting crRNAconditions (first row) and Gluc-targeting crRNA conditions (second row).(E) Pairwise comparisons of individual replicates of non-targeting shRNAconditions against the Gluc-targeting shRNA conditions. (F) Pairwisecomparisons of individual replicates of non-targeting crRNA conditionsagainst the Gluc-targeting crRNA conditions.

FIG. 61A-61C: Detailed analysis of LwaCas13a and RNAi knockdownvariability (standard deviation) across all samples. (A) Heatmap ofcorrelations (Kendall's tau) for log 2(transcripts per million (TPM+1))values of all genes detected in RNA-seq libraries between targeting andnon-targeting replicates for shRNA or crRNA targeting either luciferasereporters or endogenous genes. (B) Heatmap of correlations (Kendall'stau) for log 2(transcripts per million (TPM+1)) values of all genesdetected in RNA-seq libraries between all replicates and perturbations.(C) Distributions of standard deviations for log 2(transcripts permillion (TPM+1)) values of all genes detected in RNA-seq libraries amongtargeting and non-targeting replicates for each gene targeted for eithershRNA or crRNA.

FIG. 62A-62J: LwaCas13a knockdown is specific to the targeted transcriptwith no activity on a measured off-target transcript. (A) Heatmap ofabsolute Gluc signal for first 96 spacers tiling Gluc. (B) Heatmap ofabsolute Cluc signal for first 96 spacers tiling Gluc. (C) Relationshipbetween absolute Gluc signal and normalized luciferase for Gluc tilingguides. (D) Relationship between absolute Cluc signal and normalizedluciferase for Gluc tiling guides. (E) Relationship between absoluteCluc signal and normalized luciferase for Cluc tiling guides. (F)Relationship between absolute Gluc signal and normalized luciferase forCluc tiling guides. (G) Relationship between PPIB 2-Ct levels and PPIBknockdown for PPIB tiling guides. (H) Relationship between GAPDH 2-Ctlevels and PPIB knockdown for PPIB tiling guides. (I) Relationshipbetween KRAS 2-Ct levels and KRAS knockdown for PPIB KRAS guides. (J)Relationship between GAPDH 2-Ct levels and KRAS knockdown for PPIB KRASguides.

FIG. 63A-63F: dCas13a represses reporter gene expression and bindsendogenous genes. (A) dCas13a tiled across a synthetic HBG1 intronseparating Cluc and Gluc is capable of repressing Gluc translation atspecific distances from the translation initiation site. (B) RNAimmunoprecipitation enrichment of the β-actin mRNA targeted with dCas13aand two targeting crRNAs and one non-targeting crRNA. (C) Comparisonbetween localization of dCas13-GFP and dCas13a-GFP-KRAB constructs forimaging ACTB. (D) Additional fields of view of the dCas13a-NLS-msfGFPnegative-feedback construct delivered with a non-targeting guide. (E)Additional fields of view of the dCas13a-NLS-msfGFP negative-feedbackconstruct delivered with ACTB guide. (F) Additional fields of view ofthe dCas13a-NLS-msfGFP negative-feedback construct delivered with ACTBguide.

FIG. 64A-64B dCas13a-NF can image stress granule formation in livingcells. (A) Representative images from RNA FISH of the ACTB transcript indCas13a-NF-expressing cells with corresponding ACTB-targeting andnon-targeting guides. Cell outline is shown with a dashed line. (B)Overall signal overlap between ACTB RNA FISH signal and dCas13a-NFquantified by the Mander's overlap coefficient (left) and Pearson'scorrelation (right). Correlations and signal overlap are calculatedpixel-by-pixel on a per cell basis. All values are mean±SEM with n=3.****p<0.0001; ***p<0.001; **p<0.01. A two-tailed student's T-test wasused for comparisons.

FIG. 65A-65C. Direct and collateral transcript knockdown of expression.(A) Knockdown of luciferase by active v. dead Cas13a. (B) Active vs deadCas13a knockdown of endogenous gene expression. Knockdown by dead Cas13acan be due to blocking of translation or destabilizing of transcriptsdue to binding. (C) Absence of collateral activity by dead Cas13a inmammalian cells. Using dead Cas13a and guides 1 and 2 against luciferasein (A), no change in the transcript distribution size is observedcompared to a non-targeting control.

FIG. 66A-66O. Alignment of sequences of different C2C2 orthologs of FIG.53 (SEQ ID NOS: 68, 69, 59-61, 80, 62-64, 73, 65, 66, 79, 71, 425 and67, respectively, in order of appearance) with consensus sequence (SEQID NO: 180) indicated.

FIG. 67A-67F. Alignment of Leptotrichia wadei F0279 C2c2 (“Lew2C2c2”)(SEQ ID NO: 67) and Listeria newyorkensis FSL M6-0635 C2c2 (“LibC2c2”)(SEQ ID NO: 66).

FIG. 68 . Alignment of C2c2 HEPN domains, which display RNAse activity.The top alignment blocks include selected HEPN domains (SEQ ID NOS:451-474, respectively, in order of appearance) described previously andthe bottom blocks include the catalytic motifs from the C2c2 effectorproteins (SEQ ID NOS: 475-546, respectively, in order of appearance).Underneath each domain architecture, an alignment of the conservedmotifs in selected representatives of the respective protein family. Thecatalytic residues are shown by white letters on a black background;conserved hydrophobic residues are highlighted in yellow; conservedsmall residues are highlighted in green; in the bridge helix alignment,positively charged residues are in red. Secondary structure predictionis shown underneath the aligned sequences: H denotes a-helix and Edenotes extended conformation (β-strand). The poorly conserved spacersbetween the alignment blocks are shown by numbers.

The figures herein are for illustrative purposes only and are notnecessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

In general, a CRISPR-Cas or CRISPR system as used in the foregoingdocuments, such as WO 2014/093622 (PCT/US2013/074667) and referscollectively to transcripts and other elements involved in theexpression of or directing the activity of CRISPR-associated (“Cas”)genes, including sequences encoding a Cas gene, a tracr(trans-activating CRISPR) sequence (e.g. tracrRNA or an active partialtracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or “RNA(s)” as that term isherein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNAand transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimericRNA)) or other sequences and transcripts from a CRISPR locus. Ingeneral, a CRISPR system is characterized by elements that promote theformation of a CRISPR complex at the site of a target sequence (alsoreferred to as a protospacer in the context of an endogenous CRISPRsystem). When the CRISPR protein is a C2c2 protein, a tracrRNA is notrequired.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to havecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. A targetsequence may comprise RNA polynucleotides. In some embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In someembodiments, direct repeats may be identified in silico by searching forrepetitive motifs that fulfill any or all of the following criteria: 1.found in a 2 Kb window of genomic sequence flanking the type II CRISPRlocus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. Insome embodiments, 2 of these criteria may be used, for instance 1 and 2,2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a CRISPR complex to the target sequence. The term “targetingsequence” means the portion of a guide sequence having sufficientcomplementarity with a target sequence. In some embodiments, the degreeof complementarity between a guide sequence and its corresponding targetsequence, when optimally aligned using a suitable alignment algorithm,is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%,99%, or more. Optimal alignment may be determined with the use of anysuitable algorithm for aligning sequences, non-limiting example of whichinclude the Smith-Waterman algorithm, the Needleman-Wunsch algorithm,algorithms based on the Burrows-Wheeler Transform (e.g. the BurrowsWheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (NovocraftTechnologies; available at www.novocraft.com), ELAND (Illumina, SanDiego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. Preferably the guide sequence is 10 30nucleotides long. The ability of a guide sequence to directsequence-specific binding of a CRISPR complex to a target sequence maybe assessed by any suitable assay. For example, the components of aCRISPR system sufficient to form a CRISPR complex, including the guidesequence to be tested, may be provided to a host cell having thecorresponding target sequence, such as by transfection with vectorsencoding the components of the CRISPR sequence, followed by anassessment of preferential cleavage within the target sequence, such asby Surveyor assay as described herein. Similarly, cleavage of a targetpolynucleotide sequence may be evaluated in a test tube by providing thetarget sequence, components of a CRISPR complex, including the guidesequence to be tested and a control guide sequence different from thetest guide sequence, and comparing binding or rate of cleavage at thetarget sequence between the test and control guide sequence reactions.Other assays are possible, and will occur to those skilled in the art.

In a classic CRISPR-Cas systems, the degree of complementarity between aguide sequence and its corresponding target sequence can be about ormore than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%;a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA orsgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, orfewer nucleotides in length. However, an aspect of the invention is toreduce off-target interactions, e.g., reduce the guide interacting witha target sequence having low complementarity. Indeed, in the examples,it is shown that the invention involves mutations that result in theCRISPR-Cas system being able to distinguish between target andoff-target sequences that have greater than 80% to about 95%complementarity, e.g., 83%-84% or 88-89% or 94-95% complementarity (forinstance, distinguishing between a target having 18 nucleotides from anoff-target of 18 nucleotides having 1, 2 or 3 mismatches). Accordingly,in the context of the present invention the degree of complementaritybetween a guide sequence and its corresponding target sequence isgreater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90%or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80%complementarity between the sequence and the guide, with it advantageousthat off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98%or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementaritybetween the sequence and the guide.

In certain embodiments, modulations of cleavage efficiency can beexploited by introduction of mismatches, e.g. 1 or more mismatches, suchas 1 or 2 mismatches between spacer sequence and target sequence,including the position of the mismatch along the spacer/target. The morecentral (i.e. not 3′ or 5′) for instance a double mismatch is, the morecleavage efficiency is affected. Accordingly, by choosing mismatchposition along the spacer, cleavage efficiency can be modulated. Bymeans of example, if less than 100% cleavage of targets is desired (e.g.in a cell population), 1 or more, such as preferably 2 mismatchesbetween spacer and target sequence may be introduced in the spacersequences. The more central along the spacer of the mismatch position,the lower the cleavage percentage.

The methods according to the invention as described herein comprehendinducing one or more nucleotide modifications in a eukaryotic cell (invitro, i.e. in an isolated eukaryotic cell) as herein discussedcomprising delivering to cell a vector as herein discussed. Themutation(s) can include the introduction, deletion, or substitution ofone or more nucleotides at each target sequence of cell(s) via theguide(s) RNA(s) or sgRNA(s). The mutations can include the introduction,deletion, or substitution of 1-75 nucleotides at each target sequence ofsaid cell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 1, 5, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45,50, or 75 nucleotides at each target sequence of said cell(s) via theguide(s) RNA(s). The mutations can include the introduction, deletion,or substitution of 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides ateach target sequence of said cell(s) via the guide(s) RNA(s). Themutations include the introduction, deletion, or substitution of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45, 50, or 75 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s). The mutations can include theintroduction, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides at each targetsequence of said cell(s) via the guide(s) RNA(s). The mutations caninclude the introduction, deletion, or substitution of 40, 45, 50, 75,100, 200, 300, 400 or 500 nucleotides at each target sequence of saidcell(s) via the guide(s) RNA(s).

For minimization of toxicity and off-target effect, it will be importantto control the concentration of Cas mRNA or protein and guide RNAdelivered. Optimal concentrations of Cas mRNA or protein and guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing to analyzethe extent of modification at potential off-target genomic loci.

Typically, in the context of an endogenous CRISPR system, formation of aCRISPR complex (comprising a guide sequence hybridized to a targetsequence and complexed with one or more Cas proteins) results incleavage in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50,or more base pairs from) the target sequence, but may depend on forinstance secondary structure, in particular in the case of RNA targets.

The nucleic acid molecule encoding a Cas is advantageously codonoptimized Cas. An example of a codon optimized sequence, is in thisinstance a sequence optimized for expression in a eukaryote, e.g.,humans (i.e. being optimized for expression in humans), or for anothereukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 humancodon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a Cas is codon optimizedfor expression in particular cells, such as eukaryotic cells. Theeukaryotic cells may be those of or derived from a particular organism,such as a mammal, including but not limited to human, or non-humaneukaryote or animal or mammal as herein discussed, e.g., mouse, rat,rabbit, dog, livestock, or non-human mammal or primate. In someembodiments, processes for modifying the germ line genetic identity ofhuman beings and/or processes for modifying the genetic identity ofanimals which are likely to cause them suffering without any substantialmedical benefit to man or animal, and also animals resulting from suchprocesses, may be excluded. In general, codon optimization refers to aprocess of modifying a nucleic acid sequence for enhanced expression inthe host cells of interest by replacing at least one codon (e.g. aboutor more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) ofthe native sequence with codons that are more frequently or mostfrequently used in the genes of that host cell while maintaining thenative amino acid sequence. Various species exhibit particular bias forcertain codons of a particular amino acid. Codon bias (differences incodon usage between organisms) often correlates with the efficiency oftranslation of messenger RNA (mRNA), which is in turn believed to bedependent on, among other things, the properties of the codons beingtranslated and the availability of particular transfer RNA (tRNA)molecules. The predominance of selected tRNAs in a cell is generally areflection of the codons used most frequently in peptide synthesis.Accordingly, genes can be tailored for optimal gene expression in agiven organism based on codon optimization. Codon usage tables arereadily available, for example, at the “Codon Usage Database” availableat www.kazusa.or.jp/codon/ and these tables can be adapted in a numberof ways. See Nakamura, Y., et al. “Codon usage tabulated from theinternational DNA sequence databases: status for the year 2000” Nucl.Acids Res. 28:292 (2000). Computer algorithms for codon optimizing aparticular sequence for expression in a particular host cell are alsoavailable, such as Gene Forge (Aptagen; Jacobus, Pa.), are alsoavailable. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5,10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cascorrespond to the most frequently used codon for a particular aminoacid.

In certain embodiments, the methods as described herein may compriseproviding a Cas transgenic cell in which one or more nucleic acidsencoding one or more guide RNAs are provided or introduced operablyconnected in the cell with a regulatory element comprising a promoter ofone or more gene of interest. As used herein, the term “Cas transgeniccell” refers to a cell, such as a eukaryotic cell, in which a Cas genehas been genomically integrated. The nature, type, or origin of the cellare not particularly limiting according to the present invention. Alsothe way how the Cas transgene is introduced in the cell may vary and canbe any method as is known in the art. In certain embodiments, the Castransgenic cell is obtained by introducing the Cas transgene in anisolated cell. In certain other embodiments, the Cas transgenic cell isobtained by isolating cells from a Cas transgenic organism. By means ofexample, and without limitation, the Cas transgenic cell as referred toherein may be derived from a Cas transgenic eukaryote, such as a Casknock-in eukaryote. Reference is made to WO 2014/093622(PCT/US13/74667), incorporated herein by reference. Methods of US PatentPublication Nos. 20120017290 and 20110265198 assigned to SangamoBiosciences, Inc. directed to targeting the Rosa locus may be modifiedto utilize the CRISPR Cas system of the present invention. Methods of USPatent Publication No. 20130236946 assigned to Cellectis directed totargeting the Rosa locus may also be modified to utilize the CRISPR Cassystem of the present invention. By means of further example referenceis made to Platt et al. (Cell; 159(2):440-455 (2014)), describing a Cas9knock-in mouse, which is incorporated herein by reference. The Castransgene can further comprise a Lox-Stop-polyA-Lox (LSL) cassettethereby rendering Cas expression inducible by Cre recombinase.Alternatively, the Cas transgenic cell may be obtained by introducingthe Cas transgene in an isolated cell. Delivery systems for transgenesare well known in the art. By means of example, the Cas transgene may bedelivered in for instance eukaryotic cell by means of vector (e.g., AAV,adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, asalso described herein elsewhere.

It will be understood by the skilled person that the cell, such as theCas transgenic cell, as referred to herein may comprise further genomicalterations besides having an integrated Cas gene or the mutationsarising from the sequence specific action of Cas when complexed with RNAcapable of guiding Cas to a target locus, such as for instance one ormore oncogenic mutations, as for instance and without limitationdescribed in Platt et al. (2014), Chen et al., (2014) or Kumar et al.,(2009).

In some embodiments, the Cas sequence is fused to one or more nuclearlocalization sequences (NLSs) or nuclear export signals (NESs), such asabout or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs orNESs. In some embodiments, the Cas comprises about or more than about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs at or near theamino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more NLSs or NESs at or near the carboxy-terminus, or a combinationof these (e.g. zero or at least one or more NLS or NES at theamino-terminus and zero or at one or more NLS or NES at the carboxyterminus). When more than one NLS or NES is present, each may beselected independently of the others, such that a single NLS or NES maybe present in more than one copy and/or in combination with one or moreother NLSs or NESs present in one or more copies. In a preferredembodiment of the invention, the Cas comprises at most 6 NLSs. In someembodiments, an NLS or NES is considered near the N- or C-terminus whenthe nearest amino acid of the NLS or NES is within about 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptidechain from the N- or C-terminus. Non-limiting examples of NLSs includean NLS sequence derived from: the NLS of the SV40 virus large T-antigen,having the amino acid sequence PKKKRKV(SEQ ID NO: 27); the NLS fromnucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequenceKRPAATKKAGQAKKKK) (SEQ ID NO: 28); the c-myc NLS having the amino acidsequence PAAKRVKLD (SEQ ID NO: 29) or RQRRNELKRSP (SEQ ID NO: 30); thehRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 31); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 32) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 33) andPPKKARED (SEQ ID NO: 34) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 35) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 36)of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 37) and PKQKKRK (SEQID NO: 38) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ IDNO: 39) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR(SEQ ID NO: 40) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 41) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 42) of thesteroid hormone receptors (human) glucocorticoid. Non-limiting examplesof NESs include an NES sequence LYPERLRRILT (SEQ ID NO: 43)(ctgtaccctgagcggctgcggcggatcctgacc (SEQ ID NO: 44)). In general, the oneor more NLSs or NESs are of sufficient strength to drive accumulation ofthe Cas in a detectable amount in respectively the nucleus or thecytoplasm of a eukaryotic cell. In general, strength of nuclearlocalization/export activity may derive from the number of NLSs/NESs inthe Cas, the particular NLS(s) or NES(s) used, or a combination of thesefactors. Detection of accumulation in the nucleus/cytoplasm may beperformed by any suitable technique. For example, a detectable markermay be fused to the Cas, such that location within a cell may bevisualized, such as in combination with a means for detecting thelocation of the nucleus (e.g. a stain specific for the nucleus such asDAPI) or cytoplasm. Cell nuclei may also be isolated from cells, thecontents of which may then be analyzed by any suitable process fordetecting protein, such as immunohistochemistry, Western blot, or enzymeactivity assay. Accumulation in the nucleus may also be determinedindirectly, such as by an assay for the effect of CRISPR complexformation (e.g. assay for DNA cleavage or mutation at the targetsequence, or assay for altered gene expression activity affected byCRISPR complex formation and/or Cas enzyme activity), as compared to acontrol no exposed to the Cas or complex, or exposed to a Cas lackingthe one or more NLSs or NESs. In certain embodiments, other localizationtags may be fused to the Cas protein, such as without limitation forlocalizing the Cas to particular sites in a cell, such as organelles,such mitochondria, plastids, chloroplast, vesicles, golgi, (nuclear orcellular) membranes, ribosomes, nucleolus, ER, cytoskeleton, vacuoles,centrosome, nucleosome, granules, centrioles, etc.

In certain aspects the invention involves vectors, e.g. for deliveringor introducing in a cell Cas and/or RNA capable of guiding Cas to atarget locus (i.e. guide RNA), but also for propagating these components(e.g. in prokaryotic cells). A used herein, a “vector” is a tool thatallows or facilitates the transfer of an entity from one environment toanother. It is a replicon, such as a plasmid, phage, or cosmid, intowhich another DNA segment may be inserted so as to bring about thereplication of the inserted segment. Generally, a vector is capable ofreplication when associated with the proper control elements. Ingeneral, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. Vectorsinclude, but are not limited to, nucleic acid molecules that aresingle-stranded, double-stranded, or partially double-stranded; nucleicacid molecules that comprise one or more free ends, no free ends (e.g.circular); nucleic acid molecules that comprise DNA, RNA, or both; andother varieties of polynucleotides known in the art. One type of vectoris a “plasmid,” which refers to a circular double stranded DNA loop intowhich additional DNA segments can be inserted, such as by standardmolecular cloning techniques. Another type of vector is a viral vector,wherein virally-derived DNA or RNA sequences are present in the vectorfor packaging into a virus (e.g. retroviruses, replication defectiveretroviruses, adenoviruses, replication defective adenoviruses, andadeno-associated viruses (AAVs)). Viral vectors also includepolynucleotides carried by a virus for transfection into a host cell.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g. bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively-linked. Such vectors are referred to herein as “expressionvectors.” Common expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

The vector(s) can include the regulatory element(s), e.g., promoter(s).The vector(s) can comprise Cas encoding sequences, and/or a single, butpossibly also can comprise at least 3 or 8 or 16 or 32 or 48 or 50 guideRNA(s) (e.g., sgRNAs) encoding sequences, such as 1-2, 1-3, 1-4, 1-5,3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3-30, 3-32, 3-48, 3-50 RNA(s)(e.g., sgRNAs). In a single vector there can be a promoter for each RNA(e.g., sgRNA), advantageously when there are up to about 16 RNA(s); and,when a single vector provides for more than 16 RNA(s), one or morepromoter(s) can drive expression of more than one of the RNA(s), e.g.,when there are 32 RNA(s), each promoter can drive expression of twoRNA(s), and when there are 48 RNA(s), each promoter can drive expressionof three RNA(s). By simple arithmetic and well established cloningprotocols and the teachings in this disclosure one skilled in the artcan readily practice the invention as to the RNA(s) for a suitableexemplary vector such as AAV, and a suitable promoter such as the U6promoter. For example, the packaging limit of AAV is ˜4.7 kb. The lengthof a single U6-gRNA (plus restriction sites for cloning) is 361 bp.Therefore, the skilled person can readily fit about 12-16, e.g., 13U6-gRNA cassettes in a single vector. This can be assembled by anysuitable means, such as a golden gate strategy used for TALE assembly(http://www.genome-engineering.org/taleffectors/). The skilled personcan also use a tandem guide strategy to increase the number of U6-gRNAsby approximately 1.5 times, e.g., to increase from 12-16, e.g., 13 toapproximately 18-24, e.g., about 19 U6-gRNAs. Therefore, one skilled inthe art can readily reach approximately 18-24, e.g., about 19promoter-RNAs, e.g., U6-gRNAs in a single vector, e.g., an AAV vector. Afurther means for increasing the number of promoters and RNAs in avector is to use a single promoter (e.g., U6) to express an array ofRNAs separated by cleavable sequences. And an even further means forincreasing the number of promoter-RNAs in a vector, is to express anarray of promoter-RNAs separated by cleavable sequences in the intron ofa coding sequence or gene; and, in this instance it is advantageous touse a polymerase II promoter, which can have increased expression andenable the transcription of long RNA in a tissue specific manner. (see,e.g., http://nar.oxfordjoumals.org/content/34/7/e53.short,http://www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In anadvantageous embodiment, AAV may package U6 tandem gRNA targeting up toabout 50 genes. Accordingly, from the knowledge in the art and theteachings in this disclosure the skilled person can readily make and usevector(s), e.g., a single vector, expressing multiple RNAs or guidesunder the control or operatively or functionally linked to one or morepromoters-especially as to the numbers of RNAs or guides discussedherein, without any undue experimentation.

The guide RNA(s) encoding sequences and/or Cas encoding sequences, canbe functionally or operatively linked to regulatory element(s) and hencethe regulatory element(s) drive expression. The promoter(s) can beconstitutive promoter(s) and/or conditional promoter(s) and/or induciblepromoter(s) and/or tissue specific promoter(s). The promoter can beselected from the group consisting of RNA polymerases, pol I, pol II,pol III, T7, U6, H1, retroviral Rous sarcoma virus (RSV) LTR promoter,the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolatereductase promoter, the 3-actin promoter, the phosphoglycerol kinase(PGK) promoter, and the EF1α promoter. An advantageous promoter is thepromoter is U6.

Aspects of the invention relate to the identification and engineering ofnovel effector proteins associated with Class 2 CRISPR-Cas systems. In apreferred embodiment, the effector protein comprises a single-subuniteffector module. In a further embodiment the effector protein isfunctional in prokaryotic or eukaryotic cells for in vitro, in vivo orex vivo applications. An aspect of the invention encompassescomputational methods and algorithms to predict new Class 2 CRISPR-Cassystems and identify the components therein.

In one embodiment, a computational method of identifying novel Class 2CRISPR-Cas loci comprises the following steps: detecting all contigsencoding the Cas1 protein; identifying all predicted protein codinggenes within 20 kB of the cas1 gene, more particularly within the region20 kb from the start of the cas1 gene and 20 kb from the end of the cas1gene; comparing the identified genes with Cas protein-specific profilesand predicting CRISPR arrays; selecting partial and/or unclassifiedcandidate CRISPR-Cas loci containing proteins larger than 500 aminoacids (>500 aa); analyzing selected candidates using PSI-BLAST andHHpred, thereby isolating and identifying novel Class 2 CRISPR-Cas loci.In addition to the above mentioned steps, additional analysis of thecandidates may be conducted by searching metagenomics databases foradditional homologs.

In one aspect the detecting all contigs encoding the Cas1 protein isperformed by GeneMarkS which a gene prediction program as furtherdescribed in “GeneMarkS: a self-training method for prediction of genestarts in microbial genomes. Implications for finding sequence motifs inregulatory regions.” John Besemer, Alexandre Lomsadze and MarkBorodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, hereinincorporated by reference.

In one aspect the identifying all predicted protein coding genes iscarried out by comparing the identified genes with Cas protein-specificprofiles and annotating them according to NCBI Conserved Domain Database(CDD) which is a protein annotation resource that consists of acollection of well-annotated multiple sequence alignment models forancient domains and full-length proteins. These are available asposition-specific score matrices (PSSMs) for fast identification ofconserved domains in protein sequences via RPS-BLAST. CDD contentincludes NCBI-curated domains, which use 3D-structure information toexplicitly define domain boundaries and provide insights intosequence/structure/function relationships, as well as domain modelsimported from a number of external source databases (Pfam, SMART, COG,PRK, TIGRFAM). In a further aspect, CRISPR arrays were predicted using aPILER-CR program which is a public domain software for finding CRISPRrepeats as described in “PILER-CR: fast and accurate identification ofCRISPR repeats”, Edgar, R. C., BMC Bioinformatics, January 20;8:18(2007), herein incorporated by reference.

In a further aspect, the case by case analysis is performed usingPSI-BLAST (Position-Specific Iterative Basic Local Alignment SearchTool). PSI-BLAST derives a position-specific scoring matrix (PSSM) orprofile from the multiple sequence alignment of sequences detected abovea given score threshold using protein-protein BLAST. This PSSM is usedto further search the database for new matches, and is updated forsubsequent iterations with these newly detected sequences. Thus,PSI-BLAST provides a means of detecting distant relationships betweenproteins.

In another aspect, the case by case analysis is performed using HHpred,a method for sequence database searching and structure prediction thatis as easy to use as BLAST or PSI-BLAST and that is at the same timemuch more sensitive in finding remote homologs. In fact, HHpred'ssensitivity is competitive with the most powerful servers for structureprediction currently available. HHpred is the first server that is basedon the pairwise comparison of profile hidden Markov models (HMMs).Whereas most conventional sequence search methods search sequencedatabases such as UniProt or the NR, HHpred searches alignmentdatabases, like Pfam or SMART. This greatly simplifies the list of hitsto a number of sequence families instead of a clutter of singlesequences. All major publicly available profile and alignment databasesare available through HHpred. HHpred accepts a single query sequence ora multiple alignment as input. Within only a few minutes it returns thesearch results in an easy-to-read format similar to that of PSI-BLAST.Search options include local or global alignment and scoring secondarystructure similarity. HHpred can produce pairwise query-templatesequence alignments, merged query-template multiple alignments (e.g. fortransitive searches), as well as 3D structural models calculated by theMODELER software from HHpred alignments.

The term “nucleic acid-targeting system”, wherein nucleic acid is DNA orRNA, and in some aspects may also refer to DNA-RNA hybrids orderivatives thereof, refers collectively to transcripts and otherelements involved in the expression of or directing the activity of DNAor RNA-targeting CRISPR-associated (“Cas”) genes, which may includesequences encoding a DNA or RNA-targeting Cas protein and a DNA orRNA-targeting guide RNA comprising a CRISPR RNA (crRNA) sequence and (insome but not all systems) a trans-activating CRISPR/Cas system RNA(tracrRNA) sequence, or other sequences and transcripts from a DNA orRNA-targeting CRISPR locus. In general, a RNA-targeting system ischaracterized by elements that promote the formation of a DNA orRNA-targeting complex at the site of a target DNA or RNA sequence. Inthe context of formation of a DNA or RNA-targeting complex, “targetsequence” refers to a DNA or RNA sequence to which a DNA orRNA-targeting guide RNA is designed to have complementarity, wherehybridization between a target sequence and a RNA-targeting guide RNApromotes the formation of a RNA-targeting complex. In some embodiments,a target sequence is located in the nucleus or cytoplasm of a cell.

In an aspect of the invention, novel RNA targeting systems also referredto as RNA- or RNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific RNA target, inother words the enzyme can be recruited to a specific RNA target usingsaid RNA molecule.

In an aspect of the invention, novel DNA targeting systems also referredto as DNA- or DNA-targeting CRISPR/Cas or the CRISPR-Cas systemRNA-targeting system of the present application are based on identifiedType VI Cas proteins which do not require the generation of customizedproteins to target specific RNA sequences but rather a single enzyme canbe programmed by a RNA molecule to recognize a specific DNA target, inother words the enzyme can be recruited to a specific DNA target usingsaid RNA molecule.

The nucleic acids-targeting systems, the vector systems, the vectors andthe compositions described herein may be used in various nucleicacids-targeting applications, altering or modifying synthesis of a geneproduct, such as a protein, nucleic acids cleavage, nucleic acidsediting, nucleic acids splicing; trafficking of target nucleic acids,tracing of target nucleic acids, isolation of target nucleic acids,visualization of target nucleic acids, etc.

As used herein, a Cas protein or a CRISPR enzyme refers to any of theproteins presented in the new classification of CRISPR-Cas systems.

C2c2 Nuclease

The Class 2 type VI effector protein C2c2 is a RNA-guided RNase that canbe efficiently programmed to degrade ssRNA. C2c2 effector proteins ofthe invention include, without limitation, the following 21 orthologspecies including multiple CRISPR loci: Leptotrichia shahii;Leptotrichia wadei (Lw2); Listeria seeligeri; Lachnospiraceae bacteriumMA2020; Lachnospiraceae bacterium NK4A179; [Clostridium] aminophilum DSM10710; Carnobacterium gallinarum DSM 4847; Carnobacterium gallinarum DSM4847 (second CRISPR Loci); Paludibacter propionicigenes WB4; Listeriaweihenstephanensis FSL R9-0317; Listeriaceae bacterium FSL M6-0635;Leptotrichia wadei F0279; Rhodobacter capsulatus SB 1003; Rhodobactercapsulatus R121; Rhodobacter capsulatus DE442; Leptotrichia buccalisC-1013-b; Herbinix hemicellulosilytica; [Eubacterium] rectale;Eubacteriaceae bacterium CHKCI004; Blautia sp. Marseille-P2398; andLeptotrichia sp. oral taxon 879 str. F0557. Twelve (12) furthernon-limiting examples are: Lachnospiraceae bacterium NK4A144;Chloroflexus aggregans; Demequina aurantiaca; Thalassospira sp. TSL5-1;Pseudobutyrivibrio sp. OR37; Butyrivibrio sp. YAB3001; Blautia sp.Marseille-P2398; Leptotrichia sp. Marseille-P3007; Bacteroides ihuae;Porphyromonadaceae bacterium KH3CP3RA; Listeria riparia; andInsolitispirillum peregrinum.

C2c2 achieves RNA cleavage through conserved basic residues within itstwo HEPN domains. Mutation of the HEPN domain, such as (e.g. alanine)substitution of predicted HEPN domain catalytic residues can be used toconvert C2c2 into an inactive programmable RNA-binding protein (dC2c2,analogous to dCas9).

According to the invention, a consensus sequence can be generated frommultiple C2c2 orthologs, which can assist in locating conserved aminoacid residues, and motifs, including but not limited to catalyticresidues and HEPN motifs in C2c2 orthologs that mediate C2c2 function.One such consensus sequence, generated from the 33 orthologs mentionedabove using Geneious alignment is:

(SEQ ID NO: 45) MKISKVXXXVXKKXXXGKLXKXVNERNRXAKRLSNXLBKYIXXIDKIXKKEXXKKFXAXEEITLKLNQXXXBXLXKAXXDLRKDNXYSXJKKILHNEDINXEEXELLINDXLEKLXKIESXKYSYQKXXXNYXMSVQEHSKKSIXRIXESAKRNKEALDKFLKEYAXLDPRMEXLAKLRKLLELYFYFKNDXIXXEEEXNVXXHKXLKENHPDFVEXXXNKENAELNXYAIEXKKJLKYYFPXKXAKNSNDKIFEKQELKKXWIHQJENAVERILLXXGKVXYKLQXGYLAELWKIRINEIFIKYIXVGKAVAXFALRNXXKBENDILGGKIXKKLNGITSFXYEKIKAEEILQREXAVEVAFAANXLYAXDLXXIRXSILQFFGGASNWDXFLFFHFATSXISDKKWNAELIXXKKJGLVIREKLYSNNVAMFYSKDDLEKLLNXLXXFXLRASQVPSFKKVYVRXBFPQNLLKKFNDEKDDEAYSAXYYLLKEIYYNXFLPYFSANNXFFFXVKNLVLKANKDKFXXAFXDIREMNXGSPIEYLXXTQXNXXNEGRKKEEKEXDFIKFLLQIFXKGFDDYLKNNXXFILKFIPEPTEXIEIXXELQAWYIVGKFLNARKXNLLGXFXSYLKLLDDIELRALRNENIKYQSSNXEKEVLEXCLELIGLLSLDLNDYFBDEXDFAXYJGKXLDFEKKXMKDLAELXPYDQNDGENPIVNRNIXLAKKYGTLNLLEKJXDKVSEKEIKEYYELKKEIEEYXXKGEELHEEWXQXKNRVEXRDILEYXEELXGQIINYNXLXNKVLLYFQLGLHYLLLDILGRLVGYTGIWERDAXLYQIAAMYXNGLPEYIXXKKNDKYKDGQIVGXKINXFKXDKKXLYNAGLELFENXNEHKNIXIRNYIAHFNYLSKAESSLLXYSENLRXLFSYDRKLKNAVXKSLINILLRHGMVLKFKFGTDKKSVXIRSXKKIXHLKSIAKKLYYPEVXVSKEYCKLVKXLLKY K.

HEPN sequence motifs identified from the above orthologs are provided inFIGS. 49 and 50 on the basis of the first 21 orthologs and all 33orthologs respectively. Non-limiting examples of amino acid residuesthat can be mutated to generate catalytically dead C2c2 mutants, basedon the above consensus include, in or near HEPN1, D372, R377, Q/H382,and F383 or corresponding amino acids of an ortholog, and in or nearHEPN2, K893, N894, R898, N899, H903, F904, Y906, Y927, D928, K930, K932or corresponding amino acids of an ortholog.

In another non-limiting example, a sequence alignment tool to assistgeneration of a consensus sequence and identification of conservedresidues is the MUSCLE alignment tool (www.ebi.ac.uk/Tools/msa/muscle/).For example, using MUSCLE, the following amino acid locations conservedamong C2c2 orthologs can be identified in Leptotrichia wadei C2c2:K2;K5; V6; E301; L331; I335; N341; G351; K352; E375; L392; L396; D403;F446; I466; I470; R474 (HEPN); H475; H479 (HEPN), E508; P556; L561;I595; Y596; F600; Y669; I673; F681; L685; Y761; L676; L779; Y782; L836;D847; Y863; L869; I872; K879; I933; L954; I958; R961; Y965; E970; R971;D972; R1046 (HEPN), H1051 (HEPN), Y1075; D1076; K1078; K1080; I1083;11090.

FIG. 52A-K shows an alignment of C2c2 orthologs. FIG. 52L shows anexemplary sequence alignment of HEPN domains and highly conservedresidues.

C2c2 HEPN may also target DNA, or potentially DNA and/or RNA. On thebasis that the HEPN domains of C2c2 are at least capable of binding toand, in their wild-type form, cutting RNA, then it is preferred that theC2c2 effector protein has RNase function. It may also, or alternatively,have DNase function.

Thus, in some embodiments, the effector protein may be a RNA-bindingprotein, such as a dead-Cas type effector protein, which may beoptionally functionalized as described herein for instance with antranscriptional activator or repressor domain, NLS or other functionaldomain. In some embodiments, the effector protein may be a RNA-bindingprotein that cleaves a single strand of RNA. If the RNA bound is ssRNA,then the ssRNA is fully cleaved. In some embodiments, the effectorprotein may be a RNA-binding protein that cleaves a double strand ofRNA, for example if it comprises two RNase domains. If the RNA bound isdsRNA, then the dsRNA is fully cleaved.

RNase function in CRISPR systems is known, for example mRNA targetinghas been reported for certain type III CRISPR-Cas systems (Hale et al.,2014, Genes Dev, vol. 28, 2432-2443; Hale et al., 2009, Cell, vol. 139,945-956; Peng et al., 2015, Nucleic acids research, vol. 43, 406-417)and provides significant advantages. In the Staphylococcus epidermistype III-A system, transcription across targets results in cleavage ofthe target DNA and its transcripts, mediated by independent active siteswithin the Cas10-Csm ribonucleoprotein effector complex (see, Samai etal., 2015, Cell, vol. 151, 1164-1174). A CRISPR-Cas system, compositionor method targeting RNA via the present effector proteins is thusprovided.

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe effector protein at, the target site of interest on the target RNA.The target RNA may be any suitable form of RNA. This may include, insome embodiments, mRNA. In other embodiments, the target RNA may includetRNA or rRNA. In other embodiments, the target RNA may include miRNA. Inother embodiments, the target RNA may include siRNA.

Interfering RNA (RNAi) and microRNA (miRNA)

In other embodiments, the target RNA may include interfering RNA, i.e.RNA involved in an RNA interference pathway, such as shRNA, siRNA and soforth, both in eukaryotes and prokaryotes. In other embodiments, thetarget RNA may include microRNA (miRNA). Control over interfering RNA ormiRNA may help reduce off-target effects (OTE) seen with thoseapproaches by reducing the longevity of the interfering RNA or miRNA invivo or in vitro.

In certain embodiments, the target is not the miRNA itself, but themiRNA binding site of a miRNA target.

In certain embodiments, miRNAs may be sequestered (such as includingsubcellularly relocated). In certain embodiments, miRNAs may be cut,such as without limitation at hairpins.

In certain embodiments, miRNA processing (such as including turnover) isincreased or decreased.

If the effector protein and suitable guide are selectively expressed(for example spatially or temporally under the control of a suitablepromoter, for example a tissue- or cell cycle-specific promoter and/orenhancer) then this could be used to ‘protect’ the cells or systems (invivo or in vitro) from RNAi in those cells. This may be useful inneighbouring tissues or cells where RNAi is not required or for thepurposes of comparison of the cells or tissues where the effectorprotein and suitable guide are and are not expressed (i.e. where theRNAi is not controlled and where it is, respectively). The effectorprotein may be used to control or bind to molecules comprising orconsisting of RNA, such as ribozymes, ribosomes or riboswitches. Inembodiments of the invention, the RNA guide can recruit the effectorprotein to these molecules so that the effector protein is able to bindto them.

The protein system of the invention can be applied in areas of RNAitechnologies, without undue experimentation, from this disclosure,including therapeutic, assay and other applications (see, e.g., Guidi etal., PLoS Negl Trop Dis 9(5): e0003801. doi:10.1371/journal.pntd; Crottyet al., In vivo RNAi screens: concepts and applications. Shane Crotty .. . 2015 Elsevier Ltd. Published by Elsevier Inc., PesticideBiochemistry and Physiology (Impact Factor: 2.01). 01/2015; 120. DOI:10.1016/j.pestbp.2015.01.002 and Makkonen et al., Viruses 2015, 7(4),2099-2125; doi:10.3390/v7042099), because the present applicationprovides the foundation for informed engineering of the system.

Ribosomal RNA (rRNA)

For example, azalide antibiotics such as azithromycin, are well known.They target and disrupt the 50S ribosomal subunit. The present effectorprotein, together with a suitable guide RNA to target the 50S ribosomalsubunit, may be, in some embodiments, recruited to and bind to the 50Sribosomal subunit. Thus, the present effector protein in concert with asuitable guide directed at a ribosomal (especially the 50S ribosomalsubunit) target is provided. Use of this use effector protein in concertwith the suitable guide directed at the ribosomal (especially the 50sribosomal subunit) target may include antibiotic use. In particular, theantibiotic use is analogous to the action of azalide antibiotics, suchas azithromycin. In some embodiments, prokaryotic ribosomal subunits,such as the 70S subunit in prokaryotes, the 50S subunit mentioned above,the 30S subunit, as well as the 16S and 5S subunits may be targeted. Inother embodiments, eukaryotic ribosomal subunits, such as the 80Ssubunit in eukaryotes, the 60S subunit, the 40S subunit, as well as the28S, 18S. 5.8S and 5S subunits may be targeted.

In some embodiments, the effector protein may be a RNA-binding protein,optionally functionalized, as described herein. In some embodiments, theeffector protein may be a RNA-binding protein that cleaves a singlestrand of RNA. In either case, but particularly where the RNA-bindingprotein cleaves a single strand of RNA, then ribosomal function may bemodulated and, in particular, reduced or destroyed. This may apply toany ribosomal RNA and any ribosomal subunit and the sequences of rRNAare well known.

Control of ribosomal activity is thus envisaged through use of thepresent effector protein in concert with a suitable guide to theribosomal target. This may be through cleavage of, or binding to, theribosome. In particular, reduction of ribosomal activity is envisaged.This may be useful in assaying ribosomal function in vivo or in vitro,but also as a means of controlling therapies based on ribosomalactivity, in vivo or in vitro. Furthermore, control (i.e. reduction) ofprotein synthesis in an in vivo or in vitro system is envisaged, suchcontrol including antibiotic and research and diagnostic use.

Riboswitches

A riboswitch (also known as an aptazyme) is a regulatory segment of amessenger RNA molecule that binds a small molecule. This typicallyresults in a change in production of the proteins encoded by the mRNA.Thus, control of riboswitch activity is thus envisaged through use ofthe present effector protein in concert with a suitable guide to theriboswitch target. This may be through cleavage of, or binding to, theriboswitch. In particular, reduction of riboswitch activity isenvisaged. This may be useful in assaying riboswitch function in vivo orin vitro, but also as a means of controlling therapies based onriboswitch activity, in vivo or in vitro. Furthermore, control (i.e.reduction) of protein synthesis in an in vivo or in vitro system isenvisaged. This control, as for rRNA may include antibiotic and researchand diagnostic use.

Ribozymes

Ribozymes are RNA molecules having catalytic properties, analogous toenzymes (which are of course proteins). As ribozymes, both naturallyoccurring and engineered, comprise or consist of RNA, they may also betargeted by the present RNA-binding effector protein. In someembodiments, the effector protein may be a RNA-binding protein cleavesthe ribozyme to thereby disable it. Control of ribozymal activity isthus envisaged through use of the present effector protein in concertwith a suitable guide to the ribozymal target. This may be throughcleavage of, or binding to, the ribozyme. In particular, reduction ofribozymal activity is envisaged. This may be useful in assayingribozymal function in vivo or in vitro, but also as a means ofcontrolling therapies based on ribozymal activity, in vivo or in vitro.

Gene Expression, Including RNA Processing

The effector protein may also be used, together with a suitable guide,to target gene expression, including via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing, via targeting of RNApol;viral replication (in particular of satellite viruses, bacteriophagesand retroviruses, such as HBV, HBC and HIV and others listed herein)including viroids in plants; and tRNA biosynthesis. The effector proteinand suitable guide may also be used to control RNA activation (RNAa).RNAa leads to the promotion of gene expression, so control of geneexpression may be achieved that way through disruption or reduction ofRNAa and thus less promotion of gene expression. This is discussed morein detail below.

RNAi Screens

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. Control may also be exerted over orduring these screens by use of the effector protein and suitable guideto remove or reduce the activity of the RNAi in the screen and thusreinstate the activity of the (previously interfered with) gene product(by removing or reducing the interference/repression).

Satellite RNAs (satRNAs) and satellite viruses may also be treated.

Control herein with reference to RNase activity generally meansreduction, negative disruption or known-down or knock out.

In Vivo RNA Applications

Inhibition of Gene Expression

The target-specific RNAses provided herein allow for very specificcutting of a target RNA. The interference at RNA level allows formodulation both spatially and temporally and in a non-invasive way, asthe genome is not modified.

A number of diseases have been demonstrated to be treatable by mRNAtargeting. While most of these studies relate to administration ofsiRNA, it is clear that the RNA targeting effector proteins providedherein can be applied in a similar way.

Examples of mRNA targets (and corresponding disease treatments) areVEGF, VEGF-R1 and RTP801 (in the treatment of AMD and/or DME), Caspase 2(in the treatment of Naion) ADRB2 (in the treatment of intraocularpressure), TRPV1 (in the treatment of Dry eye syndrome), Syk kinase (inthe treatment of asthma), Apo B (in the treatment ofhypercholesterolemia or hypobetalipoproteinemia), PLK1, KSP and VEGF (inthe treatment of solid tumors), BCR-ABL (in the treatment of CML)(Burnett and Rossi Chem Biol. 2012, 19(1): 60-71)). Similarly, RNAtargeting has been demonstrated to be effective in the treatment ofRNA-virus mediated diseases such as HIV (targeting of HIV Tet and Rev),RSV (targeting of RSV nucleocapsid) and HCV (targeting of miR-122)(Burnett and Rossi Chem Biol. 2012, 19(1): 60-71).

It is further envisaged that the RNA targeting effector protein of theinvention can be used for mutation specific or allele specificknockdown. Guide RNA's can be designed that specifically target asequence in the transcribed mRNA comprising a mutation or anallele-specific sequence. Such specific knockdown is particularlysuitable for therapeutic applications relating to disorders associatedwith mutated or allele-specific gene products. For example, most casesof familial hypobetalipoproteinemia (FHBL) are caused by mutations inthe ApoB gene. This gene encodes two versions of the apolipoprotein Bprotein: a short version (ApoB-48) and a longer version (ApoB-100).Several ApoB gene mutations that lead to FHBL cause both versions ofApoB to be abnormally short. Specifically targeting and knockdown ofmutated ApoB mRNA transcripts with an RNA targeting effector protein ofthe invention may be beneficial in treatment of FHBL. As anotherexample, Huntington's disease (HD) is caused by an expansion of CAGtriplet repeats in the gene coding for the Huntingtin protein, whichresults in an abnormal protein. Specifically targeting and knockdown ofmutated or allele-specific mRNA transcripts encoding the Huntingtinprotein with an RNA targeting effector protein of the invention may bebeneficial in treatment of HD.

It is noted that in this context, and more generally for the variousapplications as described herein, the use of a split version of the RNAtargeting effector protein can be envisaged. Indeed, this may not onlyallow increased specificity but may also be advantageous for delivery.The C2c2 is split in the sense that the two parts of the C2c2 enzymesubstantially comprise a functioning C2c2. Ideally, the split shouldalways be so that the catalytic domain(s) are unaffected. That C2c2 mayfunction as a nuclease or it may be a dead-C2c2 which is essentially anRNA-binding protein with very little or no catalytic activity, due totypically mutation(s) in its catalytic domains.

Each half of the split C2c2 may be fused to a dimerization partner. Bymeans of example, and without limitation, employing rapamycin sensitivedimerization domains, allows to generate a chemically inducible splitC2c2 for temporal control of C2c2 activity. C2c2 can thus be renderedchemically inducible by being split into two fragments and thatrapamycin-sensitive dimerization domains may be used for controlledreassembly of the C2c2. The two parts of the split C2c2 can be thoughtof as the N′ terminal part and the C′ terminal part of the split C2c2.The fusion is typically at the split point of the C2c2. In other words,the C′ terminal of the N′ terminal part of the split C2c2 is fused toone of the dimer halves, whilst the N′ terminal of the C′ terminal partis fused to the other dimer half.

The C2c2 does not have to be split in the sense that the break is newlycreated. The split point is typically designed in silico and cloned intothe constructs. Together, the two parts of the split C2c2, the N′terminal and C′ terminal parts, form a full C2c2, comprising preferablyat least 70% or more of the wildtype amino acids (or nucleotidesencoding them), preferably at least 80% or more, preferably at least 90%or more, preferably at least 95% or more, and most preferably at least99% or more of the wildtype amino acids (or nucleotides encoding them).Some trimming may be possible, and mutants are envisaged. Non-functionaldomains may be removed entirely. What is important is that the two partsmay be brought together and that the desired C2c2 function is restoredor reconstituted. The dimer may be a homodimer or a heterodimer.

In certain embodiments, the C2c2 effector as described herein may beused for mutation-specific, or allele-specific targeting, such as. formutation-specific, or allele-specific knockdown.

The RNA targeting effector protein can moreover be fused to anotherfunctional RNAse domain, such as a non-specific RNase or Argonaute 2,which acts in synergy to increase the RNAse activity or to ensurefurther degradation of the message.

Modulation of Gene Expression Through Modulation of RNA Function

Apart from a direct effect on gene expression through cleavage of themRNA, RNA targeting can also be used to impact specific aspects of theRNA processing within the cell, which may allow a more subtle modulationof gene expression. Generally, modulation can for instance be mediatedby interfering with binding of proteins to the RNA, such as for instanceblocking binding of proteins, or recruiting RNA binding proteins.Indeed, modulations can be ensured at different levels such as splicing,transport, localization, translation and turnover of the mRNA. Similarlyin the context of therapy, it can be envisaged to address (pathogenic)malfunctioning at each of these levels by using RNA-specific targetingmolecules. In these embodiments it is in many cases preferred that theRNA targeting protein is a “dead” C2c2 that has lost the ability to cutthe RNA target but maintains its ability to bind thereto, such as themutated forms of c2c2 described herein.

A) Alternative Splicing

Many of the human genes express multiple mRNAs as a result ofalternative splicing. Different diseases have been shown to be linked toaberrant splicing leading to loss of function or gain of function of theexpressed gene. While some of these diseases are caused by mutationsthat cause splicing defects, a number of these are not. One therapeuticoption is to target the splicing mechanism directly. The RNA targetingeffector proteins described herein can for instance be used to block orpromote slicing, include or exclude exons and influence the expressionof specific isoforms and/or stimulate the expression of alternativeprotein products. Such applications are described in more detail below.

A RNA targeting effector protein binding to a target RNA can stericallyblock access of splicing factors to the RNA sequence. The RNA targetingeffector protein targeted to a splice site may block splicing at thesite, optionally redirecting splicing to an adjacent site. For instancea RNA targeting effector protein binding to the 5′ splice site bindingcan block the recruitment of the U1 component of the spliceosome,favoring the skipping of that exon. Alternatively, a RNA targetingeffector protein targeted to a splicing enhancer or silencer can preventbinding of transacting regulatory splicing factors at the target siteand effectively block or promote splicing. Exon exclusion can further beachieved by recruitment of ILF2/3 to precursor mRNA near an exon by anRNA targeting effector protein as described herein. As yet anotherexample, a glycine rich domain can be attached for recruitment of hnRNPA1 and exon exclusion (Del Gatto-Konczak et al. Mol Cell Biol. 1999January; 19(1):251-60).

In certain embodiments, through appropriate selection of gRNA, specificsplice variants may be targeted, while other splice variants will not betargeted.

In some cases the RNA targeting effector protein can be used to promoteslicing (e.g. where splicing is defective). For instance a RNA targetingeffector protein can be associated with an effector capable ofstabilizing a splicing regulatory stem-loop in order to furthersplicing. The RNA targeting effector protein can be linked to aconsensus binding site sequence for a specific splicing factor in orderto recruit the protein to the target DNA.

Examples of diseases which have been associated with aberrant splicinginclude, but are not limited to Paraneoplastic Opsoclonus MyoclonusAtaxia (or POMA), resulting from a loss of Nova proteins which regulatesplicing of proteins that function in the synapse, and Cystic Fibrosis,which is caused by defective splicing of a cystic fibrosis transmembraneconductance regulator, resulting in the production of nonfunctionalchloride channels. In other diseases aberrant RNA splicing results ingain-of-function. This is the case for instance in myotonic dystrophywhich is caused by a CUG triplet-repeat expansion (from 50 to >1500repeats) in the 3′ UTR of an mRNA, causing splicing defects.

The RNA targeting effector protein can be used to include an exon byrecruiting a splicing factor (such as U1) to a 5′splicing site topromote excision of introns around a desired exon. Such recruitmentcould be mediated trough a fusion with an arginine/serine rich domain,which functions as splicing activator (Gravely B R and Maniatis T, MolCell. 1998 (5):765-71).

It is envisaged that the RNA targeting effector protein can be used toblock the splicing machinery at a desired locus, resulting in preventingexon recognition and the expression of a different protein product. Anexample of a disorder that may treated is Duchenne muscular dystrophy(DMD), which is caused by mutations in the gene encoding for thedystrophin protein. Almost all DMD mutations lead to frameshifts,resulting in impaired dystrophin translation. The RNA targeting effectorprotein can be paired with splice junctions or exonic splicing enhancers(ESEs) thereby preventing exon recognition, resulting in the translationof a partially functional protein. This converts the lethal Duchennephenotype into the less severe Becker phenotype.

B) RNA Modification

RNA editing is a natural process whereby the diversity of gene productsof a given sequence is increased by minor modification in the RNA.Typically, the modification involves the conversion of adenosine (A) toinosine (I), resulting in an RNA sequence which is different from thatencoded by the genome. RNA modification is generally ensured by the ADARenzyme, whereby the pre-RNA target forms an imperfect duplex RNA bybase-pairing between the exon that contains the adenosine to be editedand an intronic non-coding element. A classic example of A-I editing isthe glutamate receptor GluR-B mRNA, whereby the change results inmodified conductance properties of the channel (Higuchi M, et al. Cell.1993; 75:1361-70).

According to the invention, enzymatic approaches are used to inducetransitions (A<→G or C<→U changes) or transversions (any purine to anypyrimidine of vice versa) in the RNA bases of a given transcript.Transitions can be directly induced by using adenine (ADAR1/2), (APOBEC)or cytosine deaminases (AID) which convert A to I or C to U,respectively. Transversions can be indirectly induced by localizingreactive oxygen species damage to the bases of interest, which causeschemical modifications to be added to the affected bases, such as theconversion of guanine to oxo-guanine. An oxo-guanine is recognized as aT and will thus base pair with an adenine causing translation to beaffected. Proteins that can be recruited for ROS-mediated base damageinclude APEX and mini-SOG. With both approaches, these effectors can befused to a catalytically inactive C2c2 and be recruited to sites ontranscripts where these types of mutations are desired.

In humans, a heterozygous functional-null mutation in the ADARI geneleads to a skin disease, human pigmentary genodermatosis (Miyamura Y, etal. Am J Hum Genet. 2003; 73:693-9). It is envisaged that the RNAtargeting effector proteins of the present invention can be used tocorrect malfunctioning RNA modification.

It is further envisaged that RNA adenosine methylase(N(6)-methyladenosine) can be fused to the RNA targeting effectorproteins of the invention and targeted to a transcript of interest. Thismethylase causes reversible methylation, has regulatory roles and mayaffect gene expression and cell fate decisions by modulating multipleRNA-related cellular pathways (Fu et al. Nat Rev Genet. 2014;15(5):293-306).

C) Polyadenylation

Polyadenylation of an mRNA is important for nuclear transport,translation efficiency and stability of the mRNA, and all of these, aswell as the process of polyadenylation, depend on specific RBPs. Mosteukaryotic mRNAs receive a 3′ poly(A) tail of about 200 nucleotidesafter transcription. Polyadenylation involves different RNA-bindingprotein complexes which stimulate the activity of a poly(A)polymerase(Minvielle-Sebastia L et al. Curr Opin Cell Biol. 1999; 11:352-7). It isenvisaged that the RNA-targeting effector proteins provided herein canbe used to interfere with or promote the interaction between theRNA-binding proteins and RNA.

Examples of diseases which have been linked to defective proteinsinvolved in polyadenylation are oculopharyngeal muscular dystrophy(OPMD) (Brais B, et al. Nat Genet. 1998; 18:164-7).

D) RNA export

After pre-mRNA processing, the mRNA is exported from the nucleus to thecytoplasm. This is ensured by a cellular mechanism which involves thegeneration of a carrier complex, which is then translocated through thenuclear pore and releases the mRNA in the cytoplasm, with subsequentrecycling of the carrier.

Overexpression of proteins (such as TAP) which play a role in the exportof RNA has been found to increase export of transcripts that areotherwise inefficiently exported in Xenopus (Katahira J, et al. EMBO J.1999; 18:2593-609).

E) mRNA Localization

mRNA localization ensures spatially regulated protein production.Localization of transcripts to a specific region of the cell can beensured by localization elements. In particular embodiments, it isenvisaged that the effector proteins described herein can be used totarget localization elements to the RNA of interest. The effectorproteins can be designed to bind the target transcript and shuttle themto a location in the cell determined by its peptide signal tag. Moreparticularly for instance, a RNA targeting effector protein fused to oneor more nuclear localization signal (NLS) and/or one or more nuclearexport signal (NES) can be used to alter RNA localization.

Further examples of localization signals include the zipcode bindingprotein 1(ZBP1) which ensures localization of 3-actin to the cytoplasmin several asymmetric cell types, KDEL retention sequence (SEQ ID NO:47) (localization to endoplasmic reticulum), nuclear export signal(localization to cytoplasm), mitochondrial targeting signal(localization to mitochondria), peroxisomal targeting signal(localization to peroxisome) and m6A marking/YTHDF2 (localization top-bodies). Other approaches that are envisaged are fusion of the RNAtargeting effector protein with proteins of known localization (forinstance membrane, synapse).

Alternatively, the effector protein according to the invention may forinstance be used in localization-dependent knockdown. By fusing theeffector protein to a appropriate localization signal, the effector istargeted to a particular cellular compartment. Only target RNAs residingin this compartment will effectively be targeted, whereas otherwiseidentical targets, but residing in a different cellular compartment willnot be targeted, such that a localization dependent knockdown can beestablished.

F) Translation

The RNA targeting effector proteins described herein can be used toenhance or repress translation. It is envisaged that upregulatingtranslation is a very robust way to control cellular circuits. Further,for functional studies a protein translation screen can be favorableover transcriptional upregulation screens, which have the shortcomingthat upregulation of transcript does not translate into increasedprotein production.

It is envisaged that the RNA targeting effector proteins describedherein can be used to bring translation initiation factors, such asEIF4G in the vicinity of the 5′ untranslated repeat (5′ UTR) of amessenger RNA of interest to drive translation (as described in DeGregorio et al. EMBO J. 1999; 18(17):4865-74 for a non-reprogrammableRNA binding protein). As another example GLD2, a cytoplasmic poly(A)polymerase, can be recruited to the target mRNA by an RNA targetingeffector protein. This would allow for directed polyadenylation of thetarget mRNA thereby stimulating translation.

Similarly, the RNA targeting effector proteins envisaged herein can beused to block translational repressors of mRNA, such as ZBP1(Huttelmaier S, et al. Nature. 2005; 438:512-5). By binding totranslation initiation site of a target RNA, translation can be directlyaffected.

In addition, fusing the RNA targeting effector proteins to a proteinthat stabilizes mRNAs, e.g. by preventing degradation thereof such asRNase inhibitors, it is possible to increase protein production from thetranscripts of interest.

It is envisaged that the RNA targeting effector proteins describedherein can be used to repress translation by binding in the 5′UTRregions of a RNA transcript and preventing the ribosome from forming andbeginning translation.

Further, the RNA targeting effector protein can be used to recruit Caf1,a component of the CCR4-NOT deadenylase complex, to the target mRNA,resulting in deadenylation or the target transcript and inhibition ofprotein translation.

For instance, the RNA targeting effector protein of the invention can beused to increase or decrease translation of therapeutically relevantproteins. Examples of therapeutic applications wherein the RNA targetingeffector protein can be used to downregulate or upregulate translationare in amyotrophic lateral sclerosis (ALS) and cardiovascular disorders.Reduced levels of the glial glutamate transporter EAAT2 have beenreported in ALS motor cortex and spinal cord, as well as multipleabnormal EAAT2 mRNA transcripts in ALS brain tissue. Loss of the EAAT2protein and function thought to be the main cause of excitotoxicity inALS. Restoration of EAAT2 protein levels and function may providetherapeutic benefit. Hence, the RNA targeting effector protein can bebeneficially used to upregulate the expression of EAAT2 protein, e.g. byblocking translational repressors or stabilizing mRNA as describedabove. Apolipoprotein A1 is the major protein component of high densitylipoprotein (HDL) and ApoA1 and HDL are generally considered asatheroprotective. It is envisages that the RNA targeting effectorprotein can be beneficially used to upregulate the expression of ApoA1,e.g. by blocking translational repressors or stabilizing mRNA asdescribed above.

G) mRNA Turnover

Translation is tightly coupled to mRNA turnover and regulated mRNAstability. Specific proteins have been described to be involved in thestability of transcripts (such as the ELAV/Hu proteins in neurons, KeeneJ D, 1999, Proc Natl Acad Sci USA. 96:5-7) and tristetraprolin (TTP).These proteins stabilize target mRNAs by protecting the messages fromdegradation in the cytoplasm (Peng S S et al., 1988, EMBO J.17:3461-70).

It can be envisaged that the RNA-targeting effector proteins of thepresent invention can be used to interfere with or to promote theactivity of proteins acting to stabilize mRNA transcripts, such thatmRNA turnover is affected. For instance, recruitment of human TTP to thetarget RNA using the RNA targeting effector protein would allow foradenylate-uridylate-rich element (AU-rich element) mediatedtranslational repression and target degradation. AU-rich elements arefound in the 3′ UTR of many mRNAs that code for proto-oncogenes, nucleartranscription factors, and cytokines and promote RNA stability. Asanother example, the RNA targeting effector protein can be fused to HuR,another mRNA stabilization protein (Hinman M N and Lou H, Cell Mol LifeSci 2008; 65:3168-81), and recruit it to a target transcript to prolongits lifetime or stabilize short-lived mRNA.

It is further envisaged that the RNA-targeting effector proteinsdescribed herein can be used to promote degradation of targettranscripts. For instance, m6A methyltransferase can be recruited to thetarget transcript to localize the transcript to P-bodies leading todegradation of the target.

As yet another example, an RNA targeting effector protein as describedherein can be fused to the non-specific endonuclease domain PilTN-terminus (PIN), to recruit it to a target transcript and allowdegradation thereof.

Patients with paraneoplastic neurological disorder (PND)-associatedencephalomyelitis and neuropathy are patients who develop autoantibodiesagainst Hu-proteins in tumors outside of the central nervous system(Szabo A et al. 1991, Cell.; 67:325-33) which then cross the blood-brainbarrier. It can be envisaged that the RNA-targeting effector proteins ofthe present invention can be used to interfere with the binding ofauto-antibodies to mRNA transcripts.

Patients with myotonic dystrophy type 1 (DM1), caused by the expansionof (CUG)n in the 3′ UTR of dystrophia myotonica-protein kinase (DMPK)gene, are characterized by the accumulation of such transcripts in thenucleus. It is envisaged that the RNA targeting effector proteins of theinvention fused with an endonuclease targeted to the (CUG)n repeatscould inhibit such accumulation of aberrant transcripts.

H) Interaction with Multi-Functional Proteins

Some RNA-binding proteins bind to multiple sites on numerous RNAs tofunction in diverse processes. For instance, the hnRNP A1 protein hasbeen found to bind exonic splicing silencer sequences, antagonizing thesplicing factors, associate with telomere ends (thereby stimulatingtelomere activity) and bind miRNA to facilitate Drosha-mediatedprocessing thereby affecting maturation. It is envisaged that theRNA-binding effector proteins of the present invention can interferewith the binding of RNA-binding proteins at one or more locations.

I) RNA Folding

RNA adopts a defined structure in order to perform its biologicalactivities. Transitions in conformation among alternative tertiarystructures are critical to most RNA-mediated processes. However, RNAfolding can be associated with several problems. For instance, RNA mayhave a tendency to fold into, and be upheld in, improper alternativeconformations and/or the correct tertiary structure may not besufficiently thermodynamically favored over alternative structures. TheRNA targeting effector protein, in particular a cleavage-deficient ordead RNA targeting protein, of the invention may be used to directfolding of (m)RNA and/or capture the correct tertiary structure thereof.

Use of RNA-Targeting Effector Protein in Modulating Cellular Status

In certain embodiments C2c2 in a complex with crRNA is activated uponbinding to target RNA and subsequently cleaves any nearby ssRNA targets(i.e. “collateral” or “bystander” effects). C2c2, once primed by thecognate target, can cleave other (non-complementary) RNA molecules. Suchpromiscuous RNA cleavage could potentially cause cellular toxicity, orotherwise affect cellular physiology or cell status.

Accordingly, in certain embodiments, the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein are used for or are for use in induction of cell dormancy. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell cycle arrest. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in reduction of cell growth and/or cell proliferation. Incertain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of cell anergy. In certainembodiments, the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein are used for orare for use in induction of cell apoptosis. In certain embodiments, thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein are used for or are for use ininduction of cell necrosis. In certain embodiments, the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein are used for or are for use in induction of celldeath. In certain embodiments, the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein areused for or are for use in induction of programmed cell death.

In certain embodiments, the invention relates to a method for inductionof cell dormancy comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell cycle arrest comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. In certain embodiments,the invention relates to a method for reduction of cell growth and/orcell proliferation comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell anergy comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of cell apoptosis comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein. Incertain embodiments, the invention relates to a method for induction ofcell necrosis comprising introducing or inducing the non-naturallyoccurring or engineered composition, vector system, or delivery systemsas described herein. In certain embodiments, the invention relates to amethod for induction of cell death comprising introducing or inducingthe non-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates to a method for induction of programmed cell deathcomprising introducing or inducing the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein.

The methods and uses as described herein may be therapeutic orprophylactic and may target particular cells, cell (sub)populations, orcell/tissue types. In particular, the methods and uses as describedherein may be therapeutic or prophylactic and may target particularcells, cell (sub)populations, or cell/tissue types expressing one ormore target sequences, such as one or more particular target RNA (e.g.ssRNA). Without limitation, target cells may for instance be cancercells expressing a particular transcript, e.g. neurons of a given class,(immune) cells causing e.g. autoimmunity, or cells infected by aspecific (e.g. viral) pathogen, etc.

Accordingly, in certain embodiments, the invention relates to a methodfor treating a pathological condition characterized by the presence ofundesirable cells (host cells), comprising introducing or inducing thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein. In certain embodiments, theinvention relates the use of the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein fortreating a pathological condition characterized by the presence ofundesirable cells (host cells). In certain embodiments, the inventionrelates the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein for use in treating apathological condition characterized by the presence of undesirablecells (host cells). It is to be understood that preferably theCRISPR-Cas system targets a target specific for the undesirable cells.In certain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for use in treating, preventing, oralleviating cancer. In certain embodiments, the invention relates to amethod for treating, preventing, or alleviating cancer comprisingintroducing or inducing the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein. Itis to be understood that preferably the CRISPR-Cas system targets atarget specific for the cancer cells. In certain embodiments, theinvention relates to the use of the non-naturally occurring orengineered composition, vector system, or delivery systems as describedherein for treating, preventing, or alleviating infection of cells by apathogen. In certain embodiments, the invention relates to thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for use in treating, preventing, oralleviating infection of cells by a pathogen. In certain embodiments,the invention relates to a method for treating, preventing, oralleviating infection of cells by a pathogen comprising introducing orinducing the non-naturally occurring or engineered composition, vectorsystem, or delivery systems as described herein. It is to be understoodthat preferably the CRISPR-Cas system targets a target specific for thecells infected by the pathogen (e.g. a pathogen derived target). Incertain embodiments, the invention relates to the use of thenon-naturally occurring or engineered composition, vector system, ordelivery systems as described herein for treating, preventing, oralleviating an autoimmune disorder. In certain embodiments, theinvention relates to the non-naturally occurring or engineeredcomposition, vector system, or delivery systems as described herein foruse in treating, preventing, or alleviating an autoimmune disorder. Incertain embodiments, the invention relates to a method for treating,preventing, or alleviating an autoimmune disorder comprising introducingor inducing the non-naturally occurring or engineered composition,vector system, or delivery systems as described herein. It is to beunderstood that preferably the CRISPR-Cas system targets a targetspecific for the cells responsible for the autoimmune disorder (e.g.specific immune cells).

Use of RNA-Targeting Effector Protein in RNA Detection or ProteinDetection

It is further envisaged that the RNA targeting effector protein can beused for detection of nucleic acids or proteins in a biological sample.The samples can be cellular or cell-free.

In Northern blot assays. Northern blotting involves the use ofelectrophoresis to separate RNA samples by size. The RNA targetingeffector protein can be used to specifically bind and detect the targetRNA sequence.

A RNA targeting effector protein can also be fused to a fluorescentprotein (such as GFP) and used to track RNA localization in livingcells. More particularly, the RNA targeting effector protein can beinactivated in that it no longer cleaves RNA. In particular embodiments,it is envisaged that a split RNA targeting effector protein can be used,whereby the signal is dependent on the binding of both subproteins, inorder to ensure a more precise visualization. Alternatively, a splitfluorescent protein can be used that is reconstituted when multiple RNAtargeting effector protein complexes bind to the target transcript. Itis further envisaged that a transcript is targeted at multiple bindingsites along the mRNA so the fluorescent signal can amplify the truesignal and allow for focal identification. As yet another alternative,the fluorescent protein can be reconstituted form a split intein.

RNA targeting effector proteins are for instance suitably used todetermine the localization of the RNA or specific splice variants, thelevel of mRNA transcript, up- or down regulation of transcripts anddisease-specific diagnosis. The RNA targeting effector proteins can beused for visualization of RNA in (living) cells using e.g. fluorescentmicroscopy or flow cytometry, such as fluorescence-activated cellsorting (FACS) which allows for high-throughput screening of cells andrecovery of living cells following cell sorting. Further, expressionlevels of different transcripts can be assessed simultaneously understress, e.g. inhibition of cancer growth using molecular inhibitors orhypoxic conditions on cells. Another application would be to tracklocalization of transcripts to synaptic connections during a neuralstimulus using two photon microscopy.

In certain embodiments, the components or complexes according to theinvention as described herein can be used in multiplexed error-robustfluorescence in situ hybridization (MERFISH; Chen et al. Science; 2015;348(6233)), such as for instance with (fluorescently) labeled C2c2effectors.

In Vitro Apex Labeling

Cellular processes depend on a network of molecular interactions amongprotein, RNA, and DNA. Accurate detection of protein-DNA and protein-RNAinteractions is key to understanding such processes. In vitro proximitylabeling technology employs an affinity tag combined with e.g. aphotoactivatable probe to label polypeptides and RNAs in the vicinity ofa protein or RNA of interest in vitro. After UV irradiation thephotoactivatable group reacts with proteins and other molecules that arein close proximity to the tagged molecule, thereby labelling them.Labelled interacting molecules can subsequently be recovered andidentified. The RNA targeting effector protein of the invention can forinstance be used to target a probe to a selected RNA sequence.

These applications could also be applied in animal models for in vivoimaging of disease relevant applications or difficult-to culture celltypes.

The invention provides agents and methods for diagnosing and monitoringhealth states through non-invasive sampling of cell free RNA, includingtesting for risk and guiding RNA-targeted therapies, and is useful insetting where rapid administration of therapy is important to treatmentoutcomes. In one embodiment, the invention provides cancer detectionmethods and agents for circulating tumor RNA, including for monitoringrecurrence and/or development of common drug resistance mutations. Inanother embodiment, the invention provides detection methods and agentsfor detection and/or identification of bacterial species directly fromblood or serum to monitor, e.g., disease progression and sepsis. In anembodiment of the invention, the C2c2 proteins and derivative are usedto distinguish and diagnose common diseases such as rhinovirus or upperrespiratory tract infections from more serious infections such asbronchitis.

The invention provides methods and agents for rapid genotyping foremergency pharmacogenomics, including guidance for administration ofanticoagulants during myocardial infarction or stroke treatment basedon, e.g., VKORC1, CYP2C9, and CYP2C19 genotyping.

The invention provides agents and methods for monitoring foodcontamination by bacteria at all points along a food production anddelivery chain. In another embodiment, the invention provides forquality control and monitoring, e.g. by identification of food sourcesand determination of purity. In one non-limiting example, the inventionmay be used to identify or confirm a food sources, such as a species ofanimal meat and seafood.

In another embodiment, the invention is used in orensic determinations.For example, crime scene samples containing blood or other bodilyfluids. In an embodiment of the invention, the invention is used toidentify nucleic acid samples from fingerprints.

Use of RNA-Targeting Effector Protein in RNA Origami/In Vitro AssemblyLines—Combinatorics

RNA origami refers to nanoscale folded structures for creatingtwo-dimensional or three-dimensional structures using RNA as integratedtemplate. The folded structure is encoded in the RNA and the shape ofthe resulting RNA is thus determined by the synthesized RNA sequence(Geary, et al. 2014. Science, 345 (6198). pp. 799-804). The RNA origamimay act as scaffold for arranging other components, such as proteins,into complexes. The RNA targeting effector protein of the invention canfor instance be used to target proteins of interest to the RNA origamiusing a suitable guide RNA.

Use of RNA-Targeting Effector Protein in RNA Isolation or Purification,Enrichment or Depletion

It is further envisages that the RNA targeting effector protein whencomplexed to RNA can be used to isolate and/or purify the RNA. The RNAtargeting effector protein can for instance be fused to an affinity tagthat can be used to isolate and/or purify the RNA-RNA targeting effectorprotein complex. Such applications are for instance useful in theanalysis of gene expression profiles in cells. In particularembodiments, it can be envisaged that the RNA targeting effectorproteins can be used to target a specific noncoding RNA (ncRNA) therebyblocking its activity, providing a useful functional probe. In certainembodiments, the effector protein as described herein may be used tospecifically enrich for a particular RNA (including but not limited toincreasing stability, etc.), or alternatively to specifically deplete aparticular RNA (such as without limitation for instance particularsplice variants, isoforms, etc.).

Interrogation of lincRNA Function and Other Nuclear RNAs

Current RNA knockdown strategies such as siRNA have the disadvantagethat they are mostly limited to targeting cytosolic transcripts sincethe protein machinery is cytosolic. The advantage of a RNA targetingeffector protein of the present invention, an exogenous system that isnot essential to cell function, is that it can be used in anycompartment in the cell. By fusing a NLS signal to the RNA targetingeffector protein, it can be guided to the nucleus, allowing nuclear RNAsto be targeted. It is for instance envisaged to probe the function oflincRNAs. Long intergenic non-coding RNAs (lincRNAs) are a vastlyunderexplored area of research. Most lincRNAs have as of yet unknownfunctions which could be studies using the RNA targeting effectorprotein of the invention.

Identification of RNA Binding Proteins

Identifying proteins bound to specific RNAs can be useful forunderstanding the roles of many RNAs. For instance, many lincRNAsassociate with transcriptional and epigenetic regulators to controltranscription. Understanding what proteins bind to a given lincRNA canhelp elucidate the components in a given regulatory pathway. A RNAtargeting effector protein of the invention can be designed to recruit abiotin ligase to a specific transcript in order to label locally boundproteins with biotin. The proteins can then be pulled down and analyzedby mass spectrometry to identify them.

Assembly of Complexes on RNA and Substrate Shuttling

RNA targeting effector proteins of the invention can further be used toassemble complexes on RNA. This can be achieved by functionalizing theRNA targeting effector protein with multiple related proteins (e.g.components of a particular synthesis pathway). Alternatively, multipleRNA targeting effector proteins can be functionalized with suchdifferent related proteins and targeted to the same or adjacent targetRNA. Useful application of assembling complexes on RNA are for instancefacilitating substrate shuttling between proteins.

Synthetic Biology

The development of biological systems have a wide utility, including inclinical applications. It is envisaged that the programmable RNAtargeting effector proteins of the invention can be used fused to splitproteins of toxic domains for targeted cell death, for instance usingcancer-linked RNA as target transcript. Further, pathways involvingprotein-protein interaction can be influenced in synthetic biologicalsystems with e.g. fusion complexes with the appropriate effectors suchas kinases or other enzymes.

Protein Splicing: Inteins

Protein splicing is a post-translational process in which an interveningpolypeptide, referred to as an intein, catalyzes its own excision fromthe polypeptides flacking it, referred to as exteins, as well assubsequent ligation of the exteins. The assembly of two or more RNAtargeting effector proteins as described herein on a target transcriptcould be used to direct the release of a split intein (Topilina andMills Mob DNA. 2014 Feb. 4; 5(1):5), thereby allowing for directcomputation of the existence of a mRNA transcript and subsequent releaseof a protein product, such as a metabolic enzyme or a transcriptionfactor (for downstream actuation of transcription pathways). Thisapplication may have significant relevance in synthetic biology (seeabove) or large-scale bioproduction (only produce product under certainconditions).

Inducible, Dosed and Self-Inactivating Systems

In one embodiment, fusion complexes comprising an RNA targeting effectorprotein of the invention and an effector component are designed to beinducible, for instance light inducible or chemically inducible. Suchinducibility allows for activation of the effector component at adesired moment in time.

Light inducibility is for instance achieved by designing a fusioncomplex wherein CRY2 PHR/CIBN pairing is used for fusion. This system isparticularly useful for light induction of protein interactions inliving cells (Konermann S, et al. Nature. 2013; 500:472-476).

Chemical inducibility is for instance provided for by designing a fusioncomplex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding)pairing is used for fusion. Using this system rapamycin is required forbinding of proteins (Zetsche et al. Nat Biotechnol. 2015; 33(2):139-42describes the use of this system for Cas9).

Further, when introduced in the cell as DNA, the RNA targeting effectorprotein of the inventions can be modulated by inducible promoters, suchas tetracycline or doxycycline controlled transcriptional activation(Tet-On and Tet-Off expression system), hormone inducible geneexpression system such as for instance an ecdysone inducible geneexpression system and an arabinose-inducible gene expression system.When delivered as RNA, expression of the RNA targeting effector proteincan be modulated via a riboswitch, which can sense a small molecule liketetracycline (as described in Goldfless et al. Nucleic Acids Res. 2012;40(9):e64).

In one embodiment, the delivery of the RNA targeting effector protein ofthe invention can be modulated to change the amount of protein or crRNAin the cell, thereby changing the magnitude of the desired effect or anyundesired off-target effects.

In one embodiment, the RNA targeting effector proteins described hereincan be designed to be self-inactivating. When delivered to a cell asRNA, either mRNA or as a replication RNA therapeutic (Wrobleska et al.Nat Biotechnol. 2015 August; 33(8): 839-841), they can self-inactivateexpression and subsequent effects by destroying the own RNA, therebyreducing residency and potential undesirable effects.

For further in vivo applications of RNA targeting effector proteins asdescribed herein, reference is made to Mackay J P et al. (Nat Struct MolBiol. 2011 March; 18(3):256-61), Nelles et al. (Bioessays. 2015 July;37(7):732-9) and Abil Z and Zhao H (Mol Biosyst. 2015 October;11(10):2658-65), which are incorporated herein by reference. Inparticular, the following applications are envisaged in certainembodiments of the invention, preferably in certain embodiments by usingcatalytically inactive C2c2: enhancing translation (e.g.C2c2-translation promotion factor fusions (e.g. eIF4 fusions));repressing translation (e.g. gRNA targeting ribosome binding sites);exon skipping (e.g. gRNAs targeting splice donor and/or acceptor sites);exon inclusion (e.g. gRNA targeting a particular exon splice donorand/or acceptor site to be included or C2c2 fused to or recruitingspliceosome components (e.g. U1 snRNA)); accessing RNA localization(e.g. C2c2-marker fusions (e.g., EGFP fusions)); altering RNAlocalization (e.g. C2c2-localization signal fusions (e.g. NLS or NESfusions)); RNA degradation (in this case no catalytically inactive C2c2is to be used if relied on the activity of C2c2, alternatively and forincreased specificity, a split C2c2 may be used); inhibition ofnon-coding RNA function (e.g. miRNA), such as by degradation or bindingof gRNA to functional sites (possibly titrating out at specific sites byrelocalization by C2c2-signal sequence fusions).

As described herein before and demonstrated in the Examples, C2c2function is robust to 5′ or 3′ extensions of the crRNA and to extensionof the crRNA loop. It is therefore envisages that MS2 loops and otherrecruitment domains can be added to the crRNA without affecting complexformation and binding to target transcripts. Such modifications to thecrRNA for recruitment of various effector domains are applicable in theuses of a RNA targeted effector proteins described above.

As demonstrated in the Examples, C2c2, in particular LshC2c2, is capableof mediating resistance to RNA phages. It is therefore envisaged thatC2c2 can be used to immunize, e.g. animals, humans and plants, againstRNA-only pathogens, including but not limited to retroviruses (e.g.lentiviruses, such as HIV), HCV, Ebola virus and Zika virus.

The present inventors have shown that C2c2 can processes (cleaves) itsown array. This applies to both the wildtype C2c2 protein and themutated C2c2 protein containing one or more mutated amino acid residuesR597, H602, R1278 and H1283, such as one or more of the modificationsselected from R597A, H602A, R1278A and H1283A. It is therefore envisagedthat multiple crRNAs designed for different target transcripts and/orapplications can be delivered as a single pre-crRNA or as a singletranscript driven by one promotor. Such method of delivery has theadvantages that it is substantially more compact, easier to synthesizeand easier to delivery in viral systems. Preferably, amino acidnumbering as described herein refers to LshC2c2 protein. It will beunderstood that exact amino acid positions may vary for orthologues ofLshC2c2, which can be adequately determined by protein alignment, as isknown in the art, and as described herein elsewhere.

Aspects of the invention also encompass methods and uses of thecompositions and systems described herein in genome or transcriptomeengineering, e.g. for altering or manipulating the (protein) expressionof one or more genes or the one or more gene products, in prokaryotic oreukaryotic cells, in vitro, in vivo or ex vivo.

In an aspect, the invention provides methods and compositions formodulating, e.g., reducing, (protein) expression of a target RNA incells. In the subject methods, a C2c2 system of the invention isprovided that interferes with transcription, stability, and/ortranslation of an RNA.

In certain embodiments, an effective amount of C2c2 system is used tocleave RNA or otherwise inhibit RNA expression. In this regard, thesystem has uses similar to siRNA and shRNA, thus can also be substitutedfor such methods. The method includes, without limitation, use of a C2c2system as a substitute for e.g., an interfering ribonucleic acid (suchas an siRNA or shRNA) or a transcription template thereof, e.g., a DNAencoding an shRNA. The C2c2 system is introduced into a target cell,e.g., by being administered to a mammal that includes the target cell.

Advantageously, a C2c2 system of the invention is specific. For example,whereas interfering ribonucleic acid (such as an siRNA or shRNA)polynucleotide systems are plagued by design and stability issues andoff-target binding, a C2c2 system of the invention can be designed withhigh specificity.

Destabilized C2c2

In certain embodiments, the effector protein (CRISPR enzyme; C2c2)according to the invention as described herein is associated with orfused to a destabilization domain (DD). In some embodiments, the DD isER50. A corresponding stabilizing ligand for this DD is, in someembodiments, 4HT. As such, in some embodiments, one of the at least oneDDs is ER50 and a stabilizing ligand therefor is 4HT or CMP8 In someembodiments, the DD is DHFR50. A corresponding stabilizing ligand forthis DD is, in some embodiments, TMP. As such, in some embodiments, oneof the at least one DDs is DHFR50 and a stabilizing ligand therefor isTMP. In some embodiments, the DD is ER50. A corresponding stabilizingligand for this DD is, in some embodiments, CMP8. CMP8 may therefore bean alternative stabilizing ligand to 4HT in the ER50 system. While itmay be possible that CMP8 and 4HT can/should be used in a competitivematter, some cell types may be more susceptible to one or the other ofthese two ligands, and from this disclosure and the knowledge in the artthe skilled person can use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminal endof the CRISPR enzyme with one or two DDs fused to the C-terminal of theCRISPR enzyme. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are the same DD, i.e. the DDs arehomologous. Thus, both (or two or more) of the DDs could be ER50 DDs.This is preferred in some embodiments. Alternatively, both (or two ormore) of the DDs could be DHFR50 DDs. This is also preferred in someembodiments. In some embodiments, the at least two DDs are associatedwith the CRISPR enzyme and the DDs are different DDs, i.e. the DDs areheterologous. Thus, one of the DDS could be ER50 while one or more ofthe DDs or any other DDs could be DHFR50. Having two or more DDs whichare heterologous may be advantageous as it would provide a greater levelof degradation control. A tandem fusion of more than one DD at the N- orC-terminus may enhance degradation; and such a tandem fusion can be, forexample ER50-ER50-C2c2 or DHFR-DHFR-C2c2. It is envisaged that highlevels of degradation would occur in the absence of either stabilizingligand, intermediate levels of degradation would occur in the absence ofone stabilizing ligand and the presence of the other (or another)stabilizing ligand, while low levels of degradation would occur in thepresence of both (or two of more) of the stabilizing ligands. Controlmay also be imparted by having an N-terminal ER50 DD and a C-terminalDHFR50 DD.

In some embodiments, the fusion of the CRISPR enzyme with the DDcomprises a linker between the DD and the CRISPR enzyme. In someembodiments, the linker is a Gly-Ser linker. In some embodiments, theDD-CRISPR enzyme further comprises at least one Nuclear Export Signal(NES). In some embodiments, the DD-CRISPR enzyme comprises two or moreNESs. In some embodiments, the DD-CRISPR enzyme comprises at least oneNuclear Localization Signal (NLS). This may be in addition to an NES. Insome embodiments, the CRISPR enzyme comprises or consists essentially ofor consists of a localization (nuclear import or export) signal as, oras part of, the linker between the CRISPR enzyme and the DD. HA or Flagtags are also within the ambit of the invention as linkers. Applicantsuse NLS and/or NES as linker and also use Glycine Serine linkers asshort as GS up to (GGGGS)₃ (SEQ ID NO: 46).

Destabilizing domains have general utility to confer instability to awide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar. 7,2012; 134(9): 3942-3945, incorporated herein by reference. CMP8 or4-hydroxytamoxifen can be destabilizing domains. More generally, atemperature-sensitive mutant of mammalian DHFR (DHFRts), a destabilizingresidue by the N-end rule, was found to be stable at a permissivetemperature but unstable at 37° C. The addition of methotrexate, ahigh-affinity ligand for mammalian DHFR, to cells expressing DHFRtsinhibited degradation of the protein partially. This was an importantdemonstration that a small molecule ligand can stabilize a proteinotherwise targeted for degradation in cells. A rapamycin derivative wasused to stabilize an unstable mutant of the FRB domain of mTOR (FRB*)and restore the function of the fused kinase, GSK-3β.^(6,7) This systemdemonstrated that ligand-dependent stability represented an attractivestrategy to regulate the function of a specific protein in a complexbiological environment. A system to control protein activity can involvethe DD becoming functional when the ubiquitin complementation occurs byrapamycin induced dimerization of FK506-binding protein and FKBP12.Mutants of human FKBP12 or ecDHFR protein can be engineered to bemetabolically unstable in the absence of their high-affinity ligands,Shield-1 or trimethoprim (TMP), respectively. These mutants are some ofthe possible destabilizing domains (DDs) useful in the practice of theinvention and instability of a DD as a fusion with a CRISPR enzymeconfers to the CRISPR protein degradation of the entire fusion proteinby the proteasome. Shield-1 and TMP bind to and stabilize the DD in adose-dependent manner. The estrogen receptor ligand binding domain(ERLBD, residues 305-549 of ERS1) can also be engineered as adestabilizing domain. Since the estrogen receptor signaling pathway isinvolved in a variety of diseases such as breast cancer, the pathway hasbeen widely studied and numerous agonist and antagonists of estrogenreceptor have been developed. Thus, compatible pairs of ERLBD and drugsare known. There are ligands that bind to mutant but not wild-type formsof the ERLBD. By using one of these mutant domains encoding threemutations (L384M, M421G, G521R)¹², it is possible to regulate thestability of an ERLBD-derived DD using a ligand that does not perturbendogenous estrogen-sensitive networks. An additional mutation (Y537S)can be introduced to further destabilize the ERLBD and to configure itas a potential DD candidate. This tetra-mutant is an advantageous DDdevelopment. The mutant ERLBD can be fused to a CRISPR enzyme and itsstability can be regulated or perturbed using a ligand, whereby theCRISPR enzyme has a DD. Another DD can be a 12-kDa (107-amino-acid) tagbased on a mutated FKBP protein, stabilized by Shieldi ligand; see,e.g., Nature Methods 5, (2008). For instance a D D can be a modifiedFK506 binding protein 12 (FKBP12) that binds to and is reversiblystabilized by a synthetic, biologically inert small molecule, Shield-1;see, e.g., Banaszynski L A, Chen L C, Maynard-Smith L A, Ooi A G,Wandless T J. A rapid, reversible, and tunable method to regulateprotein function in living cells using synthetic small molecules. Cell.2006; 126:995-1004; Banaszynski L A, Sellmyer M A, Contag C H, WandlessT J, Thorne S H. Chemical control of protein stability and function inliving mice. Nat Med. 2008; 14:1123-1127; Maynard-Smith L A, Chen L C,Banaszynski L A, Ooi A G, Wandless T J. A directed approach forengineering conditional protein stability using biologically silentsmall molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar. 23, 2012; 19(3):391-398-all of which are incorporated herein by reference and may beemployed in the practice of the invention in selected a DD to associatewith a CRISPR enzyme in the practice of this invention. As can be seen,the knowledge in the art includes a number of DDs, and the DD can beassociated with, e.g., fused to, advantageously with a linker, to aCRISPR enzyme, whereby the DD can be stabilized in the presence of aligand and when there is the absence thereof the DD can becomedestabilized, whereby the CRISPR enzyme is entirely destabilized, or theDD can be stabilized in the absence of a ligand and when the ligand ispresent the DD can become destabilized; the DD allows the CRISPR enzymeand hence the CRISPR-Cas complex or system to be regulated or controlledturned on or off so to speak, to thereby provide means for regulation orcontrol of the system, e.g., in an in vivo or in vitro environment. Forinstance, when a protein of interest is expressed as a fusion with theDD tag, it is destabilized and rapidly degraded in the cell, e.g., byproteasomes. Thus, absence of stabilizing ligand leads to a D associatedCas being degraded. When a new DD is fused to a protein of interest, itsinstability is conferred to the protein of interest, resulting in therapid degradation of the entire fusion protein. Peak activity for Cas issometimes beneficial to reduce off-target effects. Thus, short bursts ofhigh activity are preferred. The present invention is able to providesuch peaks. In some senses the system is inducible. In some othersenses, the system repressed in the absence of stabilizing ligand andde-repressed in the presence of stabilizing ligand.

Application of RNA Targeting RNA Targeting-CRISPR System to Plants andYeast

Definitions

In general, the term “plant” relates to any various photosynthetic,eukaryotic, unicellular or multicellular organism of the kingdom Plantaecharacteristically growing by cell division, containing chloroplasts,and having cell walls comprised of cellulose. The term plant encompassesmonocotyledonous and dicotyledonous plants. Specifically, the plants areintended to comprise without limitation angiosperm and gymnosperm plantssuch as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash tree,asparagus, avocado, banana, barley, beans, beet, birch, beech,blackberry, blueberry, broccoli, Brussel's sprouts, cabbage, canola,cantaloupe, carrot, cassava, cauliflower, cedar, a cereal, celery,chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee,corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive,eucalyptus, fennel, figs, fir, geranium, grape, grapefruit, groundnuts,ground cherry, gum hemlock, hickory, kale, kiwifruit, kohlrabi, larch,lettuce, leek, lemon, lime, locust, pine, maidenhair, maize, mango,maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm,okra, onion, orange, an ornamental plant or flower or tree, papaya,palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper,persimmon, pigeon pea, pine, pineapple, plantain, plum, pomegranate,potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye,sorghum, safflower, sallow, soybean, spinach, spruce, squash,strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn,tangerine, tea, tobacco, tomato, trees, triticale, turf grasses,turnips, vine, walnut, watercress, watermelon, wheat, yams, yew, andzucchini. The term plant also encompasses Algae, which are mainlyphotoautotrophs unified primarily by their lack of roots, leaves andother organs that characterize higher plants.

The methods for modulating gene expression using the RNA targeting RNAtargeting system as described herein can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g. Arabidopsis). Thus, the methods and CRISPR-Cas systemscan be used over a broad range of plants, such as for example withdicotyledonous plants belonging to the orders Magnoliales, Illiciales,Laurales, Piperales, Aristolochiales, Nymphaeales, Ranunculales,Papaverines, Sarraceniaceae, Trochodendrales, Hamarnelidales,Eucommiales, Leitneriales, Myricales, Fagales, Casuarinales,CaryophylIales, Batales, Polygonales, Plumbaginales, Dilleniales,Theales, Malvales, Urticales, Lecythidales, Violales, Salicales,Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales,Fabales, Podostemales, -Laloragales, Myrtales, Cornales, Proteales,Santalales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,Sapindales, Juglandales, Geraniales, Polygalales, UnmbelIales,Gentianales, Polernoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales; the methods andCRISPR-Cas systems can be used with rnonocotyledonous plants such asthose belonging to the orders Alismatales, Hydrocharitales, Najadales,Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales,Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales,Pandanales, Arales, Liliales and Orchid ales, or with plants belongingto Gymnospermae, e.g those belonging to the orders Pinales, Ginkgoales,Cycadales, Araucariales, Cupressales and Gnetales.

The RNA targeting CRISPR systems and methods of use described herein canbe used over a broad range of plant species, included in thenon-limitative list of dicot, monocot or gymnosperm genera hereunder:Atropa, AUseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica,Cartharnus, Cocculus, Croton, Cucunis, Citrus, Citrulus, Capsicum,Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia,Ficus, Fragaria, Glaucium, Glycine, Gossyphm, Helicanthus, Hevea,Hyosvyainus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus,MaJinihot, Majorcina, Mlalus, Medicago, Nicotiana, Olea, Parthenium,Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus,Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobromwa,Trifolium, Trigonella, Vicia, Vinca, Vitis, and Vigna; and the generaAllium, Andropogon, Aragrostis, Asparagus, Avena, Cvnodon, Elaeis,Festuca, Festulolium, Hemerocalhs, Hordeumn, Lemna, Lohim, Musa, Oryza,Panicum, Pennisetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies,Cunninghamia, Ephedra, Picea, Pinus, and Pseudotsuga.

The RNA targeting CRISPR systems and methods of use can also be usedover a broad range of “algae” or “algae cells”; including for examplealgae selected from several eukaryotic phyla, including the Rhodophyta(red algae), Chlorophyta (green algae), Phaeophyta (brown algae),Bacillariophyta (diatoms), Eustigmatophytes and dinoflagellates as wellas the prokaryotic phylum Cyanobacteria (blue-green algae). The term“algae” includes for example algae selected from: Amphora, Anabaena,Ankistrodesmus, Botryococcus, Chaetoceros, Chlamydomionas, Chlorella,Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliania, Euglena,Haematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris,Nannochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia,Nodularia, Nostoc, Ochromonas, Oocystis, Oscillatoria, Pavlova,Phaeodactylum, Platymonas, Pleurochrysis, Porphyra, Pseudoanabaena,Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis,Thalassiosira, and Trichodesmium.

A part of a plant, i.e., a “plant tissue” may be treated according tothe methods of the present invention to produce an improved plant. Planttissue also encompasses plant cells. The term “plant cell” as usedherein refers to individual units of a living plant, either in an intactwhole plant or in an isolated form grown in in vitro tissue cultures, onmedia or agar, in suspension in a growth media or buffer or as a part ofhigher organized unites, such as, for example, plant tissue, a plantorgan, or a whole plant.

A “protoplast” refers to a plant cell that has had its protective cellwall completely or partially removed using, for example, mechanical orenzymatic means resulting in an intact biochemical competent unit ofliving plant that can reform their cell wall, proliferate and regenerategrow into a whole plant under proper growing conditions.

The term “transformation” broadly refers to the process by which a planthost is genetically modified by the introduction of DNA by means ofAgrobacteria or one of a variety of chemical or physical methods. Asused herein, the term “plant host” refers to plants, including anycells, tissues, organs, or progeny of the plants. Many suitable planttissues or plant cells can be transformed and include, but are notlimited to, protoplasts, somatic embryos, pollen, leaves, seedlings,stems, calli, stolons, microtubers, and shoots. A plant tissue alsorefers to any clone of such a plant, seed, progeny, propagule whethergenerated sexually or asexually, and descendants of any of these, suchas cuttings or seed.

The term “transformed” as used herein, refers to a cell, tissue, organ,or organism into which a foreign DNA molecule, such as a construct, hasbeen introduced. The introduced DNA molecule may be integrated into thegenomic DNA of the recipient cell, tissue, organ, or organism such thatthe introduced DNA molecule is transmitted to the subsequent progeny. Inthese embodiments, the “transformed” or “transgenic” cell or plant mayalso include progeny of the cell or plant and progeny produced from abreeding program employing such a transformed plant as a parent in across and exhibiting an altered phenotype resulting from the presence ofthe introduced DNA molecule. Preferably, the transgenic plant is fertileand capable of transmitting the introduced DNA to progeny through sexualreproduction.

The term “progeny”, such as the progeny of a transgenic plant, is onethat is born of, begotten by, or derived from a plant or the transgenicplant. The introduced DNA molecule may also be transiently introducedinto the recipient cell such that the introduced DNA molecule is notinherited by subsequent progeny and thus not considered “transgenic”.Accordingly, as used herein, a “non-transgenic” plant or plant cell is aplant which does not contain a foreign DNA stably integrated into itsgenome.

The term “plant promoter” as used herein is a promoter capable ofinitiating transcription in plant cells, whether or not its origin is aplant cell. Exemplary suitable plant promoters include, but are notlimited to, those that are obtained from plants, plant viruses, andbacteria such as Agrobacterium or Rhizobium which comprise genesexpressed in plant cells.

As used herein, a “fungal cell” refers to any type of eukaryotic cellwithin the kingdom of fungi. Phyla within the kingdom of fungi includeAscomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota,Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cellsmay include yeasts, molds, and filamentous fungi. In some embodiments,the fungal cell is a yeast cell.

As used herein, the term “yeast cell” refers to any fungal cell withinthe phyla Ascomycota and Basidiomycota. Yeast cells may include buddingyeast cells, fission yeast cells, and mold cells. Without being limitedto these organisms, many types of yeast used in laboratory andindustrial settings are part of the phylum Ascomycota. In someembodiments, the yeast cell is an S. cerevisiae, Kluyveromycesmarxianus, or Issatchenkia orientalis cell. Other yeast cells mayinclude without limitation Candida spp. (e.g., Candida albicans),Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichiapastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis andKluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa),Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g.,Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candidaacidothermophilum). In some embodiments, the fungal cell is afilamentous fungal cell. As used herein, the term “filamentous fungalcell” refers to any type of fungal cell that grows in filaments, i.e.,hyphae or mycelia. Examples of filamentous fungal cells may includewithout limitation Aspergillus spp. (e.g., Aspergillus niger),Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g.,Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As usedherein, “industrial strain” refers to any strain of fungal cell used inor isolated from an industrial process, e.g., production of a product ona commercial or industrial scale. Industrial strain may refer to afungal species that is typically used in an industrial process, or itmay refer to an isolate of a fungal species that may be also used fornon-industrial purposes (e.g., laboratory research). Examples ofindustrial processes may include fermentation (e.g., in production offood or beverage products), distillation, biofuel production, productionof a compound, and production of a polypeptide. Examples of industrialstrains may include, without limitation, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As usedherein, a “polyploid” cell may refer to any cell whose genome is presentin more than one copy. A polyploid cell may refer to a type of cell thatis naturally found in a polyploid state, or it may refer to a cell thathas been induced to exist in a polyploid state (e.g., through specificregulation, alteration, inactivation, activation, or modification ofmeiosis, cytokinesis, or DNA replication). A polyploid cell may refer toa cell whose entire genome is polyploid, or it may refer to a cell thatis polyploid in a particular genomic locus of interest. Without wishingto be bound to theory, it is thought that the abundance of guide RNA maymore often be a rate-limiting component in genome engineering ofpolyploid cells than in haploid cells, and thus the methods using theC2c2 CRISPRs system described herein may take advantage of using acertain fungal cell type.

In some embodiments, the fungal cell is a diploid cell. As used herein,a “diploid” cell may refer to any cell whose genome is present in twocopies. A diploid cell may refer to a type of cell that is naturallyfound in a diploid state, or it may refer to a cell that has beeninduced to exist in a diploid state (e.g., through specific regulation,alteration, inactivation, activation, or modification of meiosis,cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cellmay refer to a cell whose entire genome is diploid, or it may refer to acell that is diploid in a particular genomic locus of interest. In someembodiments, the fungal cell is a haploid cell. As used herein, a“haploid” cell may refer to any cell whose genome is present in onecopy. A haploid cell may refer to a type of cell that is naturally foundin a haploid state, or it may refer to a cell that has been induced toexist in a haploid state (e.g., through specific regulation, alteration,inactivation, activation, or modification of meiosis, cytokinesis, orDNA replication). For example, the S. cerevisiae strain S228C may bemaintained in a haploid or diploid state. A haploid cell may refer to acell whose entire genome is haploid, or it may refer to a cell that ishaploid in a particular genomic locus of interest.

As used herein, a “yeast expression vector” refers to a nucleic acidthat contains one or more sequences encoding an RNA and/or polypeptideand may further contain any desired elements that control the expressionof the nucleic acid(s), as well as any elements that enable thereplication and maintenance of the expression vector inside the yeastcell. Many suitable yeast expression vectors and features thereof areknown in the art; for example, various vectors and techniques areillustrated in Yeast Protocols, 2nd edition, Xiao, W., ed. (HumanaPress, New York, 2007) and Buckholz, R. G. and Gleeson, M. A. (1991)Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, withoutlimitation, a centromeric (CEN) sequence, an autonomous replicationsequence (ARS), a promoter, such as an RNA Polymerase III promoter,operably linked to a sequence or gene of interest, a terminator such asan RNA polymerase III terminator, an origin of replication, and a markergene (e.g., auxotrophic, antibiotic, or other selectable markers).Examples of expression vectors for use in yeast may include plasmids,yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids,yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable Integration of RNA Targeting CRISP System Components in theGenome of Plants and Plant Cells

In particular embodiments, it is envisaged that the polynucleotidesencoding the components of the RNA targeting CRISPR system areintroduced for stable integration into the genome of a plant cell. Inthese embodiments, the design of the transformation vector or theexpression system can be adjusted depending on when, where and underwhat conditions the guide RNA and/or the RNA targeting gene(s) areexpressed.

In particular embodiments, it is envisaged to introduce the componentsof the RNA targeting CRISPR system stably into the genomic DNA of aplant cell. Additionally or alternatively, it is envisaged to introducethe components of the RNA targeting CRISPR system for stable integrationinto the DNA of a plant organelle such as, but not limited to a plastid,a mitochondrion or a chloroplast.

The expression system for stable integration into the genome of a plantcell may contain one or more of the following elements: a promoterelement that can be used to express the guide RNA and/or RNA targetingenzyme in a plant cell; a 5′ untranslated region to enhance expression;an intron element to further enhance expression in certain cells, suchas monocot cells; a multiple-cloning site to provide convenientrestriction sites for inserting the one or more guide RNAs and/or theRNA targeting gene sequences and other desired elements; and a 3′untranslated region to provide for efficient termination of theexpressed transcript.

The elements of the expression system may be on one or more expressionconstructs which are either circular such as a plasmid or transformationvector, or non-circular such as linear double stranded DNA.

In a particular embodiment, a RNA targeting CRISPR expression systemcomprises at least:

-   (a) a nucleotide sequence encoding a guide RNA (gRNA) that    hybridizes with a target sequence in a plant, and wherein the guide    RNA comprises a guide sequence and a direct repeat sequence, and-   (b) a nucleotide sequence encoding a RNA targeting protein,    wherein components (a) or (b) are located on the same or on    different constructs, and whereby the different nucleotide sequences    can be under control of the same or a different regulatory element    operable in a plant cell.

DNA construct(s) containing the components of the RNA targeting CRISPRsystem, and, where applicable, template sequence may be introduced intothe genome of a plant, plant part., or plant cell by a variety ofconventional techniques. The process generally comprises the steps ofselecting a suitable host cell or host tissue, introducing theconstruct(s) into the host cell or host tissue, and regenerating plantcells or plants therefrom. In particular embodiments, the DNA constructmay be introduced into the plant cell using techniques such as but notlimited to electroporation, microinjection, aerosol beam injection ofplant cell protoplasts, or the DNA constructs can be introduced directlyto plant tissue using biolistic methods, such as DNA particlebombardment (see also Fu et al., Transgenic Res. 2000 February;9(1):11-9). The basis of particle bombardment is the acceleration ofparticles coated with gene/s of interest toward cells, resulting in thepenetration of the protoplasm by the particles and typically stableintegration into the genome. (see e.g. Klein et al., Nature (1987),Klein et al., Bio/Technology (1992), Casas et al., Proc. Natl. Acad.Sci. USA (1993)).

In particular embodiments, the DNA constructs containing components ofthe RNA targeting CRISPR system may be introduced into the plant byAgrobacterium-mediated transformation. The DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The foreign DNA canbe incorporated into the genome of plants by infecting the plants or byincubating plant protoplasts with Agrobacterium bacteria, containing oneor more Ti (tumor-inducing) plasmids. (see e.g. Fraley et al., (1985),Rogers et al., (1987) and U.S. Pat. No. 5,563,055).

Plant Promoters

In order to ensure appropriate expression in a plant cell, thecomponents of the C2c2 CRISPR system described herein are typicallyplaced under control of a plant promoter, i.e. a promoter operable inplant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of the plant(referred to as “constitutive expression”). One non-limiting example ofa constitutive promoter is the cauliflower mosaic virus 35S promoter.The present invention envisages methods for modifying RNA sequences andas such also envisages regulating expression of plant biomolecules. Inparticular embodiments of the present invention it is thus advantageousto place one or more elements of the RNA targeting CRISPR system underthe control of a promoter that can be regulated. “Regulated promoter”refers to promoters that direct gene expression not constitutively, butin a temporally- and/or spatially-regulated manner, and includestissue-specific, tissue-preferred and inducible promoters. Differentpromoters may direct the expression of a gene in different tissues orcell types, or at different stages of development, or in response todifferent environmental conditions. In particular embodiments, one ormore of the RNA targeting CRISPR components are expressed under thecontrol of a constitutive promoter, such as the cauliflower mosaic virus35S promoter issue-preferred promoters can be utilized to targetenhanced expression in certain cell types within a particular planttissue, for instance vascular cells in leaves or roots or in specificcells of the seed. Examples of particular promoters for use in the RNAtargeting CRISPR system—are found in Kawamata et al., (1997) Plant CellPhysiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire etal., (1992) Plant Mol Biol 20:207-18, Kuster et al., (1995) Plant MolBiol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681-91.

Examples of promoters that are inducible and that allow forspatiotemporal control of gene editing or gene expression may use a formof energy. The form of energy may include but is not limited to soundenergy, electromagnetic radiation, chemical energy and/or thermalenergy. Examples of inducible systems include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc.), or light inducible systems(Phytochrome, LOV domains, or cryptochrome)., such as a Light InducibleTranscriptional Effector (LITE) that direct changes in transcriptionalactivity in a sequence-specific manner. The components of a lightinducible system may include a RNA targeting CRISPR enzyme, alight-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283, which is herebyincorporated by reference in its entirety.

In particular embodiments, transient or inducible expression can beachieved by using, for example, chemical-regulated promotors, i.e.whereby the application of an exogenous chemical induces geneexpression. Modulating of gene expression can also be obtained by achemical-repressible promoter, where application of the chemicalrepresses gene expression. Chemical-inducible promoters include, but arenot limited to, the maize ln2-2 promoter, activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant CellPhysiol 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294),activated by hydrophobic electrophilic compounds used as pre-emergentherbicides, and the tobacco PR-1a promoter (Ono et at, (2004) BiosciBiotechnol Biochem 68:803-7) activated by salicylic acid. Promoterswhich are regulated by antibiotics, such as tetracycline-inducible andtetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet227:229-37; U.S. Pat. Nos. 5,814,618 and 5,789,156) can also be usedherein.

Translocation to and/or Expression in Specific Plant Organelles

The expression system may comprise elements for translocation to and/orexpression in a specific plant organelle.

Chloroplast Targeting

In particular embodiments, it is envisaged that the RNA targeting CRISPRsystem is used to specifically modify expression and/or translation ofchloroplast genes or to ensure expression in the chloroplast. For thispurpose use is made of chloroplast transformation methods orcompartmentalization of the RNA targeting CRISPR components to thechloroplast. For instance, the introduction of genetic modifications inthe plastid genome can reduce biosafety issues such as gene flow throughpollen.

Methods of chloroplast transformation are known in the art and includeParticle bombardment, PEG treatment, and microinjection. Additionally,methods involving the translocation of transformation cassettes from thenuclear genome to the plastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more of the RNAtargeting CRISPR components to the plant chloroplast. This is achievedby incorporating in the expression construct a sequence encoding achloroplast transit peptide (CTP) or plastid transit peptide, operablylinked to the 5′ region of the sequence encoding the RNA targetingprotein. The CTP is removed in a processing step during translocationinto the chloroplast. Chloroplast targeting of expressed proteins iswell known to the skilled artisan (see for instance Protein Transportinto Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61:157-180). In such embodiments it is also desired to target the one ormore guide RNAs to the plant chloroplast. Methods and constructs whichcan be used for translocating guide RNA into the chloroplast by means ofa chloroplast localization sequence are described, for instance, in US20040142476, incorporated herein by reference. Such variations ofconstructs can be incorporated into the expression systems of theinvention to efficiently translocate the RNA targeting-guide RNA(s).

Introduction of Polynucleotides Encoding the CRISPR-RNA Targeting Systemin Algal Cells.

Transgenic algae (or other plants such as rape) may be particularlyuseful in the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol) or other products. These may beengineered to express or overexpress high levels of oil or alcohols foruse in the oil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, RNA targeting protein and guide RNA(s) are introduced inalgae expressed using a vector that expresses RNA targeting proteinunder the control of a constitutive promoter such as Hsp70A-Rbc S2 orBeta2-tubulin. Guide RNA is optionally delivered using a vectorcontaining T7 promoter. Alternatively, RNA targeting mRNA and in vitrotranscribed guide RNA can be delivered to algal cells. Electroporationprotocols are available to the skilled person such as the standardrecommended protocol from the GeneArt Chlamydomonas Engineering kit.

Introduction of Polynucleotides Encoding RNA Targeting Components inYeast Cells

In particular embodiments, the invention relates to the use of the RNAtargeting CRISPR system for RNA editing in yeast cells. Methods fortransforming yeast cells which can be used to introduce polynucleotidesencoding the RNA targeting CRISPR system components are well known tothe artisan and are reviewed by Kawai et al., 2010, Bioeng Bugs. 2010November-December; 1(6): 395-403. Non-limiting examples includetransformation of yeast cells by lithium acetate treatment (which mayfurther include carrier DNA and PEG treatment), bombardment or byelectroporation.

Transient Expression of RNA Targeting CRISP System Components in Plantsand Plant Cell

In particular embodiments, it is envisaged that the guide RNA and/or RNAtargeting gene are transiently expressed in the plant cell. In theseembodiments, the RNA targeting CRISPR system can ensure modification ofRNA target molecules only when both the guide RNA and the RNA targetingprotein is present in a cell, such that gene expression can further becontrolled. As the expression of the RNA targeting enzyme is transient,plants regenerated from such plant cells typically contain no foreignDNA. In particular embodiments the RNA targeting enzyme is stablyexpressed by the plant cell and the guide sequence is transientlyexpressed.

In particularly preferred embodiments, the RNA targeting CRISPR systemcomponents can be introduced in the plant cells using a plant viralvector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996; 34:299-323).In further particular embodiments, said viral vector is a vector from aDNA virus. For example, geminivirus (e.g., cabbage leaf curl virus, beanyellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maizestreak virus, tobacco leaf curl virus, or tomato golden mosaic virus) ornanovirus (e.g., Faba bean necrotic yellow virus). In other particularembodiments, said viral vector is a vector from an RNA virus. Forexample, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus),potexvirus (e.g., potato virus X), or hordeivirus (e.g., barley stripemosaic virus). The replicating genomes of plant viruses arenon-integrative vectors, which is of interest in the context of avoidingthe production of GMO plants.

In particular embodiments, the vector used for transient expression ofRNA targeting CRISPR constructs is for instance a pEAQ vector, which istailored for Agrobacterium-mediated transient expression (Sainsbury F.et al., Plant Biotechnol J. 2009 September; 7(7):682-93) in theprotoplast. Precise targeting of genomic locations was demonstratedusing a modified Cabbage Leaf Curl virus (CaLCuV) vector to expressgRNAs in stable transgenic plants expressing a CRISPR enzyme (ScientificReports 5, Article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding theguide RNA and/or the RNA targeting gene can be transiently introducedinto the plant cell. In such embodiments, the introduced double-strandedDNA fragments are provided in sufficient quantity to modify RNAmolecule(s) in the cell but do not persist after a contemplated periodof time has passed or after one or more cell divisions. Methods fordirect DNA transfer in plants are known by the skilled artisan (see forinstance Davey et al. Plant Mol Biol. 1989 September; 13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding the RNA targetingprotein is introduced into the plant cell, which is then translated andprocessed by the host cell generating the protein in sufficient quantityto modify the RNA molecule(s) cell (in the presence of at least oneguide RNA) but which does not persist after a contemplated period oftime has passed or after one or more cell divisions. Methods forintroducing mRNA to plant protoplasts for transient expression are knownby the skilled artisan (see for instance in Gallie, Plant Cell Reports(1993), 13; 119-122). Combinations of the different methods describedabove are also envisaged.

Delivery of RNA Targeting CRISPR Components to the Plant Cell

In particular embodiments, it is of interest to deliver one or morecomponents of the RNA targeting CRISPR system directly to the plantcell. This is of interest, inter alia, for the generation ofnon-transgenic plants (see below). In particular embodiments, one ormore of the RNA targeting components is prepared outside the plant orplant cell and delivered to the cell. For instance in particularembodiments, the RNA targeting protein is prepared in vitro prior tointroduction to the plant cell. RNA targeting protein can be prepared byvarious methods known by one of skill in the art and include recombinantproduction. After expression, the RNA targeting protein is isolated,refolded if needed, purified and optionally treated to remove anypurification tags, such as a His-tag. Once crude, partially purified, ormore completely purified RNA targeting protein is obtained, the proteinmay be introduced to the plant cell.

In particular embodiments, the RNA targeting protein is mixed with guideRNA targeting the RNA of interest to form a pre-assembledribonucleoprotein.

The individual components or pre-assembled ribonucleoprotein can beintroduced into the plant cell via electroporation, by bombardment withRNA targeting-associated gene product coated particles, by chemicaltransfection or by some other means of transport across a cell membrane.For instance, transfection of a plant protoplast with a pre-assembledCRISPR ribonucleoprotein has been demonstrated to ensure targetedmodification of the plant genome (as described by Woo et al. NatureBiotechnology, 2015; DOI: 10.1038/nbt.3389). These methods can bemodified to achieve targeted modification of RNA molecules in theplants.

In particular embodiments, the RNA targeting CRISPR system componentsare introduced into the plant cells using nanoparticles. The components,either as protein or nucleic acid or in a combination thereof, can beuploaded onto or packaged in nanoparticles and applied to the plants(such as for instance described in WO 2008042156 and US 20130185823). Inparticular, embodiments of the invention comprise nanoparticles uploadedwith or packed with DNA molecule(s) encoding the RNA targeting protein,DNA molecules encoding the guide RNA and/or isolated guide RNA asdescribed in WO2015089419.

Further means of introducing one or more components of the RNA targetingCRISPR system to the plant cell is by using cell penetrating peptides(CPP). Accordingly, in particular, embodiments the invention comprisescompositions comprising a cell penetrating peptide linked to an RNAtargeting protein. In particular embodiments of the present invention,an RNA targeting protein and/or guide RNA(s) is coupled to one or moreCPPs to effectively transport them inside plant protoplasts(Ramakrishna, Genome Res. 2014 June; 24(6):1020-7 for Cas9 in humancells). In other embodiments, the RNA targeting gene and/or guide RNA(s)are encoded by one or more circular or non-circular DNA molecule(s)which are coupled to one or more CPPs for plant protoplast delivery. Theplant protoplasts are then regenerated to plant cells and further toplants. CPPs are generally described as short peptides of fewer than 35amino acids either derived from proteins or from chimeric sequenceswhich are capable of transporting biomolecules across cell membrane in areceptor independent manner. CPP can be cationic peptides, peptideshaving hydrophobic sequences, amphipathic peptides, peptides havingproline-rich and anti-microbial sequence, and chimeric or bipartitepeptides (Pooga and Langel 2005). CPPs are able to penetrate biologicalmembranes and as such trigger the movement of various biomoleculesacross cell membranes into the cytoplasm and to improve theirintracellular routing, and hence facilitate interaction of thebiomolecule with the target. Examples of CPP include amongst others:Tat, a nuclear transcriptional activator protein required for viralreplication by HIV type1, penetratin, Kaposi fibroblast growth factor(FGF) signal peptide sequence, integrin 33 signal peptide sequence;polyarginine peptide sequence, Guanine rich-molecular transporters,sweet arrow peptide, etc.

Target RNA Envisaged for Plant, Algae or Fungal Applications

The target RNA, i.e. the RNA of interest, is the RNA to be targeted bythe present invention leading to the recruitment to, and the binding ofthe RNA targeting protein at, the target site of interest on the targetRNA. The target RNA may be any suitable form of RNA. This may include,in some embodiments, mRNA. In other embodiments, the target RNA mayinclude transfer RNA (tRNA) or ribosomal RNA (rRNA). In otherembodiments the target RNA may include interfering RNA (RNAi), microRNA(miRNA), microswitches, microzymes, satellite RNAs and RNA viruses. Thetarget RNA may be located in the cytoplasm of the plant cell, or in thecell nucleus or in a plant cell organelle such as a mitochondrion,chloroplast or plastid.

In particular embodiments, the RNA targeting CRISPR system is used tocleave RNA or otherwise inhibit RNA expression.

Use of RNA Targeting CRISPR System for Modulating Plant Gene ExpressionVia RNA Modulation

The RNA targeting protein may also be used, together with a suitableguide RNA, to target gene expression, via control of RNA processing. Thecontrol of RNA processing may include RNA processing reactions such asRNA splicing, including alternative splicing or specifically targetingcertain splice variants or isoforms; viral replication, in particular ofplant viruses, including viroids in plants and tRNA biosynthesis. TheRNA targeting protein in combination with a suitable guide RNA may alsobe used to control RNA activation (RNAa). RNAa leads to the promotion ofgene expression, so control of gene expression may be achieved that waythrough disruption or reduction of RNAa and thus less promotion of geneexpression.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity in plants, in particular against RNA viruses. Theeffector protein can be targeted to the viral RNA using a suitable guideRNA selective for a selected viral RNA sequence. In particular, theeffector protein may be an active nuclease that cleaves RNA, such assingle stranded RNA. Provided is therefore the use of an RNA targetingeffector protein of the invention as an antiviral agent. Examples ofviruses that can be counteracted in this way include, but are notlimited to, Tobacco mosaic virus (TMV), Tomato spotted wilt virus(TSWV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Cauliflowermosaic virus (CaMV) (RT virus), Plum pox virus (PPV), Brome mosaic virus(BMV) and Potato virus X (PVX).

Examples of modulating RNA expression in plants, algae or fungi, as analternative of targeted gene modification are described herein further.

Of particular interest is the regulated control of gene expressionthrough regulated cleavage of mRNA. This can be achieved by placingelements of the RNA targeting under the control of regulated promotersas described herein.

Use of the RNA Targeting CRISPR System to Restore the Functionality oftRNA Molecules.

Pring et al. describe RNA editing in plant mitochondria and chloroplaststhat alters mRNA sequences to code for different proteins than the DNA.(Plant Mol. Biol. (1993) 21 (6): 1163-1170. doi:10.1007/BF00023611). Inparticular embodiments of the invention, the elements of the RNAtargeting CRISPR system specifically targeting mitochondrial andchloroplast mRNA can be introduced in a plant or plant cell to expressdifferent proteins in such plant cell organelles mimicking the processesoccurring in vivo.

Use of the RNA Targeting CRISPR System as an Alternative to RNAInterference to Inhibit RNA Expression.

The RNA targeting CRISPR system has uses similar to RNA inhibition orRNA interference, thus can also be substituted for such methods. Inparticular embodiment, the methods of the present invention include theuse of the RNA targeting CRISPR as a substitute for e.g. an interferingribonucleic acid (such as an siRNA or shRNA or a dsRNA). Examples ofinhibition of RNA expression in plants, algae or fungi as an alternativeof targeted gene modification are described herein further.

Use of the RNA Targeting CRISPR System to Control RNA Interference.

Control over interfering RNA or miRNA may help reduce off-target effects(OTE) seen with those approaches by reducing the longevity of theinterfering RNA or miRNA in vivo or in vitro. In particular embodiments,the target RNA may include interfering RNA, i.e. RNA involved in an RNAinterference pathway, such as shRNA, siRNA and so forth. In otherembodiments, the target RNA may include microRNA (miRNA) or doublestranded RNA (dsRNA).

In other particular embodiments, if the RNA targeting protein andsuitable guide RNA(s) are selectively expressed (for example spatiallyor temporally under the control of a regulated promoter, for example atissue- or cell cycle-specific promoter and/or enhancer) this can beused to ‘protect’ the cells or systems (in vivo or in vitro) from RNAiin those cells. This may be useful in neighbouring tissues or cellswhere RNAi is not required or for the purposes of comparison of thecells or tissues where the effector protein and suitable guide are andare not expressed (i.e. where the RNAi is not controlled and where itis, respectively). The RNA targeting protein may be used to control orbind to molecules comprising or consisting of RNA, such as ribozymes,ribosomes or riboswitches. In embodiments of the invention, the guideRNA can recruit the RNA targeting protein to these molecules so that theRNA targeting protein is able to bind to them.

The RNA targeting CRISPR system of the invention can be applied in areasof in-planta RNAi technologies, without undue experimentation, from thisdisclosure, including insect pest management, plant disease managementand management of herbicide resistance, as well as in plant assay andfor other applications (see, for instance Kim et al., in PesticideBiochemistry and Physiology (Impact Factor: 2.01). 01/2015; 120. DOI:10.1016/j.pestbp.2015.01.002; Sharma et al. in Academic Journals (2015),Vol. 12(18) pp. 2303-2312); Green J. M, in Pest Management Science, Vol70(9), pp 1351-1357), because the present application provides thefoundation for informed engineering of the system.

Use of RNA Targeting CRISPR System to Modify Riboswitches and ControlMetabolic Regulation in Plants, Algae and Fungi

Riboswitches (also known as aptazymes) are regulatory segments ofmessenger RNA that bind small molecules and in turn regulate geneexpression. This mechanism allows the cell to sense the intracellularconcentration of these small molecules. A particular riboswitchtypically regulates its adjacent gene by altering the transcription, thetranslation or the splicing of this gene. Thus, in particularembodiments of the present invention, control of riboswitch activity isenvisaged through the use of the RNA targeting protein in combinationwith a suitable guide RNA to target the riboswitch. This may be throughcleavage of, or binding to, the riboswitch. In particular embodiments,reduction of riboswitch activity is envisaged. Recently, a riboswitchthat binds thiamine pyrophosphate (TPP) was characterized and found toregulate thiamine biosynthesis in plants and algae. Furthermore itappears that this element is an essential regulator of primarymetabolism in plants (Bocobza and Aharoni, Plant J. 2014 August;79(4):693-703. doi: 10.1111/tpj.12540. Epub 2014 Jun. 17). TPPriboswitches are also found in certain fungi, such as in Neurosporacrassa, where it controls alternative splicing to conditionally producean Upstream Open Reading Frame (uORF), thereby affecting the expressionof downstream genes (Cheah M T et al., (2007) Nature 447 (7143):497-500. doi:10.1038/nature05769) The RNA targeting CRISPR systemdescribed herein may be used to manipulate the endogenous riboswitchactivity in plants, algae or fungi and as such alter the expression ofdownstream genes controlled by it. In particular embodiments, the RNAtargeting CRISP system may be used in assaying riboswitch function invivo or in vitro and in studying its relevance for the metabolicnetwork. In particular embodiments the RNA targeting CRISPR system maypotentially be used for engineering of riboswitches as metabolitesensors in plants and platforms for gene control.

Use of RNA Targeting CRISPR System in RNAi Screens for Plants, Algae orFungi

Identifying gene products whose knockdown is associated with phenotypicchanges, biological pathways can be interrogated and the constituentparts identified, via RNAi screens. In particular embodiments of theinvention, control may also be exerted over or during these screens byuse of the Guide 29 or Guide 30 protein and suitable guide RNA describedherein to remove or reduce the activity of the RNAi in the screen andthus reinstate the activity of the (previously interfered with) geneproduct (by removing or reducing the interference/repression).

Use of RNA Targeting Proteins for Visualization of RNA Molecules In Vivoand In Vitro

In particular embodiments, the invention provides a nucleic acid bindingsystem. In situ hybridization of RNA with complementary probes is apowerful technique. Typically fluorescent DNA oligonucleotides are usedto detect nucleic acids by hybridization. Increased efficiency has beenattained by certain modifications, such as locked nucleic acids (LNAs),but there remains a need for efficient and versatile alternatives. Assuch, labelled elements of the RNA targeting system can be used as analternative for efficient and adaptable system for in situhybridization.

Further Applications of the RNA Targeting CRISPR System in Plants andYeasts

Use of RNA Targeting CRISPR System in Biofuel Production

The term “biofuel” as used herein is an alternative fuel made from plantand plant-derived resources. Renewable biofuels can be extracted fromorganic matter whose energy has been obtained through a process ofcarbon fixation or are made through the use or conversion of biomass.This biomass can be used directly for biofuels or can be converted toconvenient energy containing substances by thermal conversion, chemicalconversion, and biochemical conversion. This biomass conversion canresult in fuel in solid, liquid, or gas form. There are two types ofbiofuels: bioethanol and biodiesel. Bioethanol is mainly produced by thesugar fermentation process of cellulose (starch), which is mostlyderived from maize and sugar cane. Biodiesel on the other hand is mainlyproduced from oil crops such as rapeseed, palm, and soybean. Biofuelsare used mainly for transportation.

Enhancing Plant Properties for Biofuel Production

In particular embodiments, the methods using the RNA targeting CRISPRsystem as described herein are used to alter the properties of the cellwall in order to facilitate access by key hydrolysing agents for a moreefficient release of sugars for fermentation. In particular embodiments,the biosynthesis of cellulose and/or lignin are modified. Cellulose isthe major component of the cell wall. The biosynthesis of cellulose andlignin are co-regulated. By reducing the proportion of lignin in a plantthe proportion of cellulose can be increased. In particular embodiments,the methods described herein are used to downregulate ligninbiosynthesis in the plant so as to increase fermentable carbohydrates.More particularly, the methods described herein are used to downregulateat least a first lignin biosynthesis gene selected from the groupconsisting of 4-coumarate 3-hydroxylase (C3H), phenylalanineammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeoyl CoA3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamylalcohol dehydrogenase (CAD), cinnamoyl CoA-reductase (CCR),4-coumarate-CoA ligase (4CL), monolignol-lignin-specificglycosyltransferase, and aldehyde dehydrogenase (ALDH) as disclosed inWO 2008064289 A2.

In particular embodiments, the methods described herein are used toproduce plant mass that produces lower levels of acetic acid duringfermentation (see also WO 2010096488).

Modifying Yeast for Biofuel Production

In particular embodiments, the RNA targeting enzyme provided herein isused for bioethanol production by recombinant micro-organisms. Forinstance, RNA targeting enzymes can be used to engineer micro-organisms,such as yeast, to generate biofuel or biopolymers from fermentablesugars and optionally to be able to degrade plant-derived lignocellulosederived from agricultural waste as a source of fermentable sugars. Moreparticularly, the invention provides methods whereby the RNA targetingCRISPR complex is used to modify the expression of endogenous genesrequired for biofuel production and/or to modify endogenous genes whymay interfere with the biofuel synthesis. More particularly the methodsinvolve stimulating the expression in a micro-organism such as a yeastof one or more nucleotide sequence encoding enzymes involved in theconversion of pyruvate to ethanol or another product of interest. Inparticular embodiments the methods ensure the stimulation of expressionof one or more enzymes which allows the micro-organism to degradecellulose, such as a cellulase. In yet further embodiments, the RNAtargeting CRISPR complex is used to suppress endogenous metabolicpathways which compete with the biofuel production pathway.

Modifying Algae and Plants for Production of Vegetable Oils or Biofuels

Transgenic algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

U.S. Pat. No. 8,945,839 describes a method for engineering Micro-Algae(Chlamydomonas reinhardtii cells) species using Cas9. Using similartools, the methods of the RNA targeting CRISPR system described hereincan be applied on Chlamydomonas species and other algae. In particularembodiments, the RNA targeting effector protein and guide RNA areintroduced in algae expressed using a vector that expresses the RNAtargeting effector protein under the control of a constitutive promotersuch as Hsp70A-Rbc S2 or Beta2-tubulin. Guide RNA will be deliveredusing a vector containing T7 promoter. Alternatively, in vitrotranscribed guide RNA can be delivered to algae cells. Electroporationprotocol follows standard recommended protocol from the GeneArtChlamydomonas Engineering kit.

Particular Applications of the RNA Targeting Enzymes in Plants

In particular embodiments, present invention can be used as a therapyfor virus removal in plant systems as it is able to cleave viral RNA.Previous studies in human systems have demonstrated the success ofutilizing CRISPR in targeting the single strand RNA virus, hepatitis C(A. Price, et al., Proc. Natl. Acad. Sci, 2015). These methods may alsobe adapted for using the RNA targeting CRISPR system in plants.

Improved Plants

The present invention also provides plants and yeast cells obtainableand obtained by the methods provided herein. The improved plantsobtained by the methods described herein may be useful in food or feedproduction through the modified expression of genes which, for instanceensure tolerance to plant pests, herbicides, drought, low or hightemperatures, excessive water, etc.

The improved plants obtained by the methods described herein, especiallycrops and algae may be useful in food or feed production throughexpression of, for instance, higher protein, carbohydrate, nutrient orvitamin levels than would normally be seen in the wildtype. In thisregard, improved plants, especially pulses and tubers are preferred.

Improved algae or other plants such as rape may be particularly usefulin the production of vegetable oils or biofuels such as alcohols(especially methanol and ethanol), for instance. These may be engineeredto express or overexpress high levels of oil or alcohols for use in theoil or biofuel industries.

The invention also provides for improved parts of a plant. Plant partsinclude, but are not limited to, leaves, stems, roots, tubers, seeds,endosperm, ovule, and pollen. Plant parts as envisaged herein may beviable, nonviable, regeneratable, and/or non-regeneratable.

It is also encompassed herein to provide plant cells and plantsgenerated according to the methods of the invention. Gametes, seeds,embryos, either zygotic or somatic, progeny or hybrids of plantscomprising the genetic modification, which are produced by traditionalbreeding methods, are also included within the scope of the presentinvention. Such plants may contain a heterologous or foreign DNAsequence inserted at or instead of a target sequence. Alternatively,such plants may contain only an alteration (mutation, deletion,insertion, substitution) in one or more nucleotides. As such, suchplants will only be different from their progenitor plants by thepresence of the particular modification.

In an embodiment of the invention, a C2c2 system is used to engineerpathogen resistant plants, for example by creating resistance againstdiseases caused by bacteria, fungi or viruses. In certain embodiments,pathogen resistance can be accomplished by engineering crops to producea C2c2 system that will be ingested by an insect pest, leading tomortality. In an embodiment of the invention, a C2c2 system is used toengineer abiotic stress tolerance. In another embodiment, a C2c2 systemis used to engineer drought stress tolerance or salt stress tolerance,or cold or heat stress tolerance. Younis et al. 2014, Int. J. Biol. Sci.10; 1150 reviewed potential targets of plant breeding methods, all ofwhich are amenable to correction or improvement through use of a C2c2system described herein. Some non-limiting target crops includeArabidopsis Zea mays is thaliana, Oryza sativa L, Prunus domestica L.,Gossypium hirsutum, Nicotiana rustica, Zea mays, Medicago sativa,Nicotiana benthamiana and Arabidopsis thaliana.

In an embodiment of the invention, a C2c2 system is used for managementof crop pests. For example, a C2c2 system operable in a crop pest can beexpressed from a plant host or transferred directly to the target, forexample using a viral vector.

In an embodiment, the invention provides a method of efficientlyproducing homozygous organisms from a heterozygous non-human startingorganism. In an embodiment, the invention is used in plant breeding. Inanother embodiment, the invention is used in animal breeding. In suchembodiments, a homozygous organism such as a plant or animal is made bypreventing or suppressing recombination by interfering with at least onetarget gene involved in double strand breaks, chromosome pairing and/orstrand exchange.

Application of the C2C2 Proteins in Optimized Functional RNA TargetingSystems

In an aspect the invention provides a system for specific delivery offunctional components to the RNA environment. This can be ensured usingthe CRISPR systems comprising the RNA targeting effector proteins of thepresent invention which allow specific targeting of different componentsto RNA. More particularly such components include activators orrepressors, such as activators or repressors of RNA translation,degradation, etc. Applications of this system are described elsewhereherein.

According to one aspect the invention provides non-naturally occurringor engineered composition comprising a guide RNA comprising a guidesequence capable of hybridizing to a target sequence in a genomic locusof interest in a cell, wherein the guide RNA is modified by theinsertion of one or more distinct RNA sequence(s) that bind an adaptorprotein. In particular embodiments, the RNA sequences may bind to two ormore adaptor proteins (e.g. aptamers), and wherein each adaptor proteinis associated with one or more functional domains. The guide RNAs of theC2c2 enzymes described herein are shown to be amenable to modificationof the guide sequence. In particular embodiments, the guide RNA ismodified by the insertion of distinct RNA sequence(s) 5′ of the directrepeat, within the direct repeat, or 3′ of the guide sequence. Whenthere is more than one functional domain, the functional domains can besame or different, e.g., two of the same or two different activators orrepressors. In an aspect the invention provides a herein-discussedcomposition, wherein the one or more functional domains are attached tothe RNA targeting enzyme so that upon binding to the target RNA thefunctional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function. In an aspectthe invention provides a herein-discussed composition, wherein thecomposition comprises a CRISPR-Cas complex having at least threefunctional domains, at least one of which is associated with the RNAtargeting enzyme and at least two of which are associated with the gRNA.

Accordingly, in an aspect the invention provides non-naturally occurringor engineered CRISPR-Cas complex composition comprising the guide RNA asherein-discussed and a CRISPR enzyme which is an RNA targeting enzyme,wherein optionally the RNA targeting enzyme comprises at least onemutation, such that the RNA targeting enzyme has no more than 5% of thenuclease activity of the enzyme not having the at least one mutation,and optionally one or more comprising at least one or more nuclearlocalization sequences. In particular embodiments, the guide RNA isadditionally or alternatively modified so as to still ensure binding ofthe RNA targeting enzyme but to prevent cleavage by the RNA targetingenzyme (as detailed elsewhere herein).

In particular embodiments, the RNA targeting enzyme is a c2c2 enzymewhich has a diminished nuclease activity of at least 97%, or 100% ascompared with the c2c2 enzyme not having the at least one mutation. Inan aspect the invention provides a herein-discussed composition, whereinthe C2c2 enzyme comprises two or more mutations. The mutations may beselected from mutations of one or more of the following amino acidresidues: R597, H602, R1278, and H1283, such as for instance one or moreof the following mutations: R597A, H602A, R1278A, and H1283A, accordingto Leptotrichia shahii c2c2 protein or a corresponding position in anortholog.

In particular embodiments, an RNA targeting system is provided asdescribed herein above comprising two or more functional domains. Inparticular embodiments, the two or more functional domains areheterologous functional domain. In particular embodiments, the systemcomprises an adaptor protein which is a fusion protein comprising afunctional domain, the fusion protein optionally comprising a linkerbetween the adaptor protein and the functional domain. In particularembodiments, the linker includes a Gly-Ser linker. Additionally oralternatively, one or more functional domains are attached to the RNAeffector protein by way of a linker, optionally a Gly-Ser linker. Inparticular embodiments, the one or more functional domains are attachedto the RNA targeting enzyme through one or both of the HEPN domains.

In an aspect the invention provides a herein-discussed composition,wherein the one or more functional domains associated with the adaptorprotein or the RNA targeting enzyme is a domain capable of activating orrepressing RNA translation. In an aspect the invention provides aherein-discussed composition, wherein at least one of the one or morefunctional domains associated with the adaptor protein have one or moreactivities comprising methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,DNA integration activity RNA cleavage activity, DNA cleavage activity ornucleic acid binding activity, or molecular switch activity or chemicalinducibility or light inducibility.

In an aspect the invention provides a herein-discussed compositioncomprising an aptamer sequence. In particular embodiments, the aptamersequence is two or more aptamer sequences specific to the same adaptorprotein. In an aspect the invention provides a herein-discussedcomposition, wherein the aptamer sequence is two or more aptamersequences specific to different adaptor protein. In an aspect theinvention provides a herein-discussed composition, wherein the adaptorprotein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34,JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5,ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Accordingly, in particular embodiments,the aptamer is selected from a binding protein specifically binding anyone of the adaptor proteins listed above. In an aspect the inventionprovides a herein-discussed composition, wherein the cell is aeukaryotic cell. In an aspect the invention provides a herein-discussedcomposition, wherein the eukaryotic cell is a mammalian cell, a plantcell or a yeast cell, whereby the mammalian cell is optionally a mousecell. In an aspect the invention provides a herein-discussedcomposition, wherein the mammalian cell is a human cell.

In an aspect the invention provides a herein above-discussed compositionwherein there is more than one gRNA, and the gRNAs target differentsequences whereby when the composition is employed, there ismultiplexing. In an aspect the invention provides a composition whereinthere is more than one gRNA modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins.

In an aspect the invention provides a herein-discussed compositionwherein one or more adaptor proteins associated with one or morefunctional domains is present and bound to the distinct RNA sequence(s)inserted into the guide RNA(s).

In an aspect the invention provides a herein-discussed compositionwherein the guide RNA is modified to have at least one non-codingfunctional loop; e.g., wherein the at least one non-coding functionalloop is repressive; for instance, wherein at least one non-codingfunctional loop comprises Alu.

In an aspect the invention provides a method for modifying geneexpression comprising the administration to a host or expression in ahost in vivo of one or more of the compositions as herein-discussed.

In an aspect the invention provides a herein-discussed method comprisingthe delivery of the composition or nucleic acid molecule(s) codingtherefor, wherein said nucleic acid molecule(s) are operatively linkedto regulatory sequence(s) and expressed in vivo. In an aspect theinvention provides a herein-discussed method wherein the expression invivo is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a mammalian cell line of cells asherein-discussed, wherein the cell line is, optionally, a human cellline or a mouse cell line. In an aspect the invention provides atransgenic mammalian model, optionally a mouse, wherein the model hasbeen transformed with a herein-discussed composition or is a progeny ofsaid transformant.

In an aspect the invention provides a nucleic acid molecule(s) encodingguide RNA or the RNA targeting CRISPR-Cas complex or the composition asherein-discussed. In an aspect the invention provides a vectorcomprising: a nucleic acid molecule encoding a guide RNA (gRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein the direct repeat ofthe gRNA is modified by the insertion of distinct RNA sequence(s) thatbind(s) to two or more adaptor proteins, and wherein each adaptorprotein is associated with one or more functional domains; or, whereinthe gRNA is modified to have at least one non-coding functional loop. Inan aspect the invention provides vector(s) comprising nucleic acidmolecule(s) encoding: non-naturally occurring or engineered CRISPR-Cascomplex composition comprising the gRNA herein-discussed, and an RNAtargeting enzyme, wherein optionally the RNA targeting enzyme comprisesat least one mutation, such that the RNA targeting enzyme has no morethan 5% of the nuclease activity of the RNA targeting enzyme not havingthe at least one mutation, and optionally one or more comprising atleast one or more nuclear localization sequences. In an aspect a vectorcan further comprise regulatory element(s) operable in a eukaryotic celloperably linked to the nucleic acid molecule encoding the guide RNA(gRNA) and/or the nucleic acid molecule encoding the RNA targetingenzyme and/or the optional nuclear localization sequence(s).

In one aspect, the invention provides a kit comprising one or more ofthe components described hereinabove. In some embodiments, the kitcomprises a vector system as described above and instructions for usingthe kit.

In an aspect the invention provides a method of screening for gain offunction (GOF) or loss of function (LOF) or for screening non-codingRNAs or potential regulatory regions (e.g. enhancers, repressors)comprising the cell line of as herein-discussed or cells of the modelherein-discussed containing or expressing the RNA targeting enzyme andintroducing a composition as herein-discussed into cells of the cellline or model, whereby the gRNA includes either an activator or arepressor, and monitoring for GOF or LOF respectively as to those cellsas to which the introduced gRNA includes an activator or as to thosecells as to which the introduced gRNA includes a repressor.

In an aspect the invention provides a library of non-naturally occurringor engineered compositions, each comprising a RNA targeting CRISPR guideRNA (gRNA) comprising a guide sequence capable of hybridizing to atarget RNA sequence of interest in a cell, an RNA targeting enzyme,wherein the RNA targeting enzyme comprises at least one mutation, suchthat the RNA targeting enzyme has no more than 5% of the nucleaseactivity of the RNA targeting enzyme not having the at least onemutation, wherein the gRNA is modified by the insertion of distinct RNAsequence(s) that bind to one or more adaptor proteins, and wherein theadaptor protein is associated with one or more functional domains,wherein the composition comprises one or more or two or more adaptorproteins, wherein the each protein is associated with one or morefunctional domains, and wherein the gRNAs comprise a genome wide librarycomprising a plurality of RNA targeting guide RNAs (gRNAs). In an aspectthe invention provides a library as herein-discussed, wherein the RNAtargeting RNA targeting enzyme has a diminished nuclease activity of atleast 97%, or 100% as compare with the RNA targeting enzyme not havingthe at least one mutation. In an aspect the invention provides a libraryas herein-discussed, wherein the adaptor protein is a fusion proteincomprising the functional domain. In an aspect the invention provides alibrary as herein discussed, wherein the gRNA is not modified by theinsertion of distinct RNA sequence(s) that bind to the one or two ormore adaptor proteins. In an aspect the invention provides a library asherein discussed, wherein the one or two or more functional domains areassociated with the RNA targeting enzyme. In an aspect the inventionprovides a library as herein discussed, wherein the cell population ofcells is a population of eukaryotic cells. In an aspect the inventionprovides a library as herein discussed, wherein the eukaryotic cell is amammalian cell, a plant cell or a yeast cell. In an aspect the inventionprovides a library as herein discussed, wherein the mammalian cell is ahuman cell. In an aspect the invention provides a library as hereindiscussed, wherein the population of cells is a population of embryonicstem (ES) cells.

In an aspect the invention provides a library as herein discussed,wherein the targeting is of about 100 or more RNA sequences. In anaspect the invention provides a library as herein discussed, wherein thetargeting is of about 1000 or more RNA sequences. In an aspect theinvention provides a library as herein discussed, wherein the targetingis of about 20,000 or more sequences. In an aspect the inventionprovides a library as herein discussed, wherein the targeting is of theentire transcriptome. In an aspect the invention provides a library asherein discussed, wherein the targeting is of a panel of targetsequences focused on a relevant or desirable pathway. In an aspect theinvention provides a library as herein discussed, wherein the pathway isan immune pathway. In an aspect the invention provides a library asherein discussed, wherein the pathway is a cell division pathway.

In one aspect, the invention provides a method of generating a modeleukaryotic cell comprising a gene with modified expression. In someembodiments, a disease gene is any gene associated an increase in therisk of having or developing a disease. In some embodiments, the methodcomprises (a) introducing one or more vectors encoding the components ofthe system described herein above into a eukaryotic cell, and (b)allowing a CRISPR complex to bind to a target polynucleotide so as tomodify expression of a gene, thereby generating a model eukaryotic cellcomprising modified gene expression.

The structural information provided herein allows for interrogation ofguide RNA interaction with the target RNA and the RNA targeting enzymepermitting engineering or alteration of guide RNA structure to optimizefunctionality of the entire RNA targeting CRISPR-Cas system. Forexample, the guide RNA may be extended, without colliding with the RNAtargeting protein by the insertion of adaptor proteins that can bind toRNA. These adaptor proteins can further recruit effector proteins orfusions which comprise one or more functional domains.

An aspect of the invention is that the above elements are comprised in asingle composition or comprised in individual compositions. Thesecompositions may advantageously be applied to a host to elicit afunctional effect on the genomic level.

The skilled person will understand that modifications to the guide RNAwhich allow for binding of the adapter+functional domain but not properpositioning of the adapter+functional domain (e.g. due to sterichindrance within the three dimensional structure of the CRISPR complex)are modifications which are not intended. The one or more modified guideRNA may be modified, by introduction of a distinct RNA sequence(s) 5′ ofthe direct repeat, within the direct repeat, or 3′ of the guidesequence.

The modified guide RNA, the inactivated RNA targeting enzyme (with orwithout functional domains), and the binding protein with one or morefunctional domains, may each individually be comprised in a compositionand administered to a host individually or collectively. Alternatively,these components may be provided in a single composition foradministration to a host. Administration to a host may be performed viaviral vectors known to the skilled person or described herein fordelivery to a host (e.g. lentiviral vector, adenoviral vector, AAVvector). As explained herein, use of different selection markers (e.g.for lentiviral gRNA selection) and concentration of gRNA (e.g. dependenton whether multiple gRNAs are used) may be advantageous for eliciting animproved effect.

Using the provided compositions, the person skilled in the art canadvantageously and specifically target single or multiple loci with thesame or different functional domains to elicit one or more genomicevents. The compositions may be applied in a wide variety of methods forscreening in libraries in cells and functional modeling in vivo (e.g.gene activation of lincRNA and identification of function;gain-of-function modeling; loss-of-function modeling; the use thecompositions of the invention to establish cell lines and transgenicanimals for optimization and screening purposes).

The current invention comprehends the use of the compositions of thecurrent invention to establish and utilize conditional or inducibleCRISPR RNA targeting events. (See, e.g., Platt et al., Cell (2014),http://dx.doi.org/10.1016/j.cell.2014.09.014, or PCT patent publicationscited herein, such as WO 2014/093622 (PCT/US2013/074667), which are notbelieved prior to the present invention or application). For example,the target cell comprises RNA targeting CRISPR enzyme conditionally orinducibly (e.g. in the form of Cre dependent constructs) and/or theadapter protein conditionally or inducibly and, on expression of avector introduced into the target cell, the vector expresses that whichinduces or gives rise to the condition of s RNA targeting enzymeexpression and/or adaptor expression in the target cell. By applying theteaching and compositions of the current invention with the known methodof creating a CRISPR complex, inducible gene expression affected byfunctional domains are also an aspect of the current invention.Alternatively, the adaptor protein may be provided as a conditional orinducible element with a conditional or inducible s RNA targeting enzymeto provide an effective model for screening purposes, whichadvantageously only requires minimal design and administration ofspecific gRNAs for a broad number of applications.

Guide RNA According to the Invention Comprising a Dead Guide Sequence

In one aspect, the invention provides guide sequences which are modifiedin a manner which allows for formation of the CRISPR complex andsuccessful binding to the target, while at the same time, not allowingfor successful nuclease activity (i.e. without nuclease activity/withoutindel activity). For matters of explanation such modified guidesequences are referred to as “dead guides” or “dead guide sequences”.These dead guides or dead guide sequences can be thought of ascatalytically inactive or conformationally inactive with regard tonuclease activity. Indeed, dead guide sequences may not sufficientlyengage in productive base pairing with respect to the ability to promotecatalytic activity or to distinguish on-target and off-target bindingactivity. Briefly, the assay involves synthesizing a CRISPR target RNAand guide RNAs comprising mismatches with the target RNA, combiningthese with the RNA targeting enzyme and analyzing cleavage based on gelsbased on the presence of bands generated by cleavage products, andquantifying cleavage based upon relative band intensities.

Hence, in a related aspect, the invention provides a non-naturallyoccurring or engineered composition RNA targeting CRISPR-Cas systemcomprising a functional RNA targeting as described herein, and guide RNA(gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNAis capable of hybridizing to a target sequence such that the RNAtargeting CRISPR-Cas system is directed to a genomic locus of interestin a cell without detectable RNA cleavage activity of a non-mutant RNAtargeting enzyme of the system. It is to be understood that any of thegRNAs according to the invention as described herein elsewhere may beused as dead gRNAs/gRNAs comprising a dead guide sequence as describedherein below. Any of the methods, products, compositions and uses asdescribed herein elsewhere is equally applicable with the deadgRNAs/gRNAs comprising a dead guide sequence as further detailed below.By means of further guidance, the following particular aspects andembodiments are provided.

The ability of a dead guide sequence to direct sequence-specific bindingof a CRISPR complex to an RNA target sequence may be assessed by anysuitable assay. For example, the components of a CRISPR systemsufficient to form a CRISPR complex, including the dead guide sequenceto be tested, may be provided to a host cell having the correspondingtarget sequence, such as by transfection with vectors encoding thecomponents of the CRISPR sequence, followed by an assessment ofpreferential cleavage within the target sequence. For instance, cleavageof a target RNA polynucleotide sequence may be evaluated in a test tubeby providing the target sequence, components of a CRISPR complex,including the dead guide sequence to be tested and a control guidesequence different from the test dead guide sequence, and comparingbinding or rate of cleavage at the target sequence between the test andcontrol guide sequence reactions. Other assays are possible, and willoccur to those skilled in the art. A dead guide sequence may be selectedto target any target sequence. In some embodiments, the target sequenceis a sequence within a genome of a cell.

As explained further herein, several structural parameters allow for aproper framework to arrive at such dead guides. Dead guide sequences aretypically shorter than respective guide sequences which result in activeRNA cleavage. In particular embodiments, dead guides are 5%, 10%, 20%,30%, 40%, 50%, shorter than respective guides directed to the same.

As explained below and known in the art, one aspect of gRNA-RNAtargeting specificity is the direct repeat sequence, which is to beappropriately linked to such guides. In particular, this implies thatthe direct repeat sequences are designed dependent on the origin of theRNA targeting enzyme. Thus, structural data available for validated deadguide sequences may be used for designing C2c2 specific equivalents.Structural similarity between, e.g., the orthologous nuclease domainsHEPN of two or more C2c2 effector proteins may be used to transferdesign equivalent dead guides. Thus, the dead guide herein may beappropriately modified in length and sequence to reflect such C2c2specific equivalents, allowing for formation of the CRISPR complex andsuccessful binding to the target RNA, while at the same time, notallowing for successful nuclease activity.

The use of dead guides in the context herein as well as the state of theart provides a surprising and unexpected platform for network biologyand/or systems biology in both in vitro, ex vivo, and in vivoapplications, allowing for multiplex gene targeting, and in particularbidirectional multiplex gene targeting. Prior to the use of dead guides,addressing multiple targets has been challenging and in some cases notpossible. With the use of dead guides, multiple targets, and thusmultiple activities, may be addressed, for example, in the same cell, inthe same animal, or in the same patient. Such multiplexing may occur atthe same time or staggered for a desired timeframe.

For example, the dead guides allow to use gRNA as a means for genetargeting, without the consequence of nuclease activity, while at thesame time providing directed means for activation or repression. GuideRNA comprising a dead guide may be modified to further include elementsin a manner which allow for activation or repression of gene activity,in particular protein adaptors (e.g. aptamers) as described hereinelsewhere allowing for functional placement of gene effectors (e.g.activators or repressors of gene activity). One example is theincorporation of aptamers, as explained herein and in the state of theart. By engineering the gRNA comprising a dead guide to incorporateprotein-interacting aptamers (Konermann et al., “Genome-scaletranscription activation by an engineered CRISPR-Cas9 complex,”doi:10.1038/nature14136, incorporated herein by reference), one mayassemble multiple distinct effector domains. Such may be modeled afternatural processes.

Thus, one aspect is a gRNA of the invention which comprises a deadguide, wherein the gRNA further comprises modifications which providefor gene activation or repression, as described herein. The dead gRNAmay comprise one or more aptamers. The aptamers may be specific to geneeffectors, gene activators or gene repressors. Alternatively, theaptamers may be specific to a protein which in turn is specific to andrecruits/binds a specific gene effector, gene activator or generepressor. If there are multiple sites for activator or repressorrecruitment, it is preferred that the sites are specific to eitheractivators or repressors. If there are multiple sites for activator orrepressor binding, the sites may be specific to the same activators orsame repressors. The sites may also be specific to different activatorsor different repressors. The effectors, activators, repressors may bepresent in the form of fusion proteins.

In an aspect, the invention provides a method of selecting a dead guideRNA targeting sequence for directing a functionalized CRISPR system to agene locus in an organism, which comprises: a) locating one or moreCRISPR motifs in the gene locus; b) analyzing the 20 nt sequencedownstream of each CRISPR motif by: i) determining the GC content of thesequence; and ii) determining whether there are off-target matches ofthe first 15 nt of the sequence in the genome of the organism; c)selecting the sequence for use in a guide RNA if the GC content of thesequence is 70% or less and no off-target matches are identified. In anembodiment, the sequence is selected if the GC content is 50% or less.In an embodiment, the sequence is selected if the GC content is 40% orless. In an embodiment, the sequence is selected if the GC content is30% or less. In an embodiment, two or more sequences are analyzed andthe sequence having the lowest GC content is selected. In an embodiment,off-target matches are determined in regulatory sequences of theorganism. In an embodiment, the gene locus is a regulatory region. Anaspect provides a dead guide RNA comprising the targeting sequenceselected according to the aforementioned methods.

In an aspect, the invention provides a dead guide RNA for targeting afunctionalized CRISPR system to a gene locus in an organism. In anembodiment of the invention, the dead guide RNA comprises a targetingsequence wherein the CG content of the target sequence is 70% or less,and the first 15 nt of the targeting sequence does not match anoff-target sequence downstream from a CRISPR motif in the regulatorysequence of another gene locus in the organism. In certain embodiments,the GC content of the targeting sequence 60% or less, 55% or less, 50%or less, 45% or less, 40% or less, 35% or less or 30% or less. Incertain embodiments, the GC content of the targeting sequence is from70% to 60% or from 60% to 50% or from 50% to 40% or from 40% to 30%. Inan embodiment, the targeting sequence has the lowest CG content amongpotential targeting sequences of the locus.

In an embodiment of the invention, the first 15 nt of the dead guidematch the target sequence. In another embodiment, first 14 nt of thedead guide match the target sequence. In another embodiment, the first13 nt of the dead guide match the target sequence. In another embodimentfirst 12 nt of the dead guide match the target sequence. In anotherembodiment, first 11 nt of the dead guide match the target sequence. Inanother embodiment, the first 10 nt of the dead guide match the targetsequence. In an embodiment of the invention the first 15 nt of the deadguide does not match an off-target sequence downstream from a CRISPRmotif in the regulatory region of another gene locus. In otherembodiments, the first 14 nt, or the first 13 nt of the dead guide, orthe first 12 nt of the guide, or the first 11 nt of the dead guide, orthe first 10 nt of the dead guide, does not match an off-target sequencedownstream from a CRISPR motif in the regulatory region of another genelocus. In other embodiments, the first 15 nt, or 14 nt, or 13 nt, or 12nt, or 11 nt of the dead guide do not match an off-target sequencedownstream from a CRISPR motif in the genome.

In certain embodiments, the dead guide RNA includes additionalnucleotides at the 3′-end that do not match the target sequence. Thus, adead guide RNA that includes the first 20-28 nt, downstream of a CRISPRmotif can be extended in length at the 3′ end.

General Provisions

In an aspect, the invention provides a nucleic acid binding system. Insitu hybridization of RNA with complementary probes is a powerfultechnique. Typically fluorescent DNA oligonucleotides are used to detectnucleic acids by hybridization. Increased efficiency has been attainedby certain modifications, such as locked nucleic acids (LNAs), but thereremains a need for efficient and versatile alternatives. The inventionprovides an efficient and adaptable system for in situ hybridization.

In embodiments of the invention the terms guide sequence and guide RNAare used interchangeably as in foregoing cited documents such as WO2014/093622 (PCT/US2013/074667). In general, a guide sequence is anypolynucleotide sequence having sufficient complementarity with a targetpolynucleotide sequence to hybridize with the target sequence and directsequence-specific binding of a CRISPR complex to the target sequence. Insome embodiments, the degree of complementarity between a guide sequenceand its corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies; available atwww.novocraft.com), ELAND (Illumina, San Diego, Calif.), SOAP (availableat soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). Insome embodiments, a guide sequence is about or more than about 5, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In someembodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30,25, 20, 15, 12, or fewer nucleotides in length. Preferably the guidesequence is 10-30 nucleotides long. The ability of a guide sequence todirect sequence-specific binding of a CRISPR complex to a targetsequence may be assessed by any suitable assay. For example, thecomponents of a CRISPR system sufficient to form a CRISPR complex,including the guide sequence to be tested, may be provided to a hostcell having the corresponding target sequence, such as by transfectionwith vectors encoding the components of the CRISPR sequence, followed byan assessment of preferential cleavage within the target sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget polynucleotide sequence may be evaluated in a test tube byproviding the target sequence, components of a CRISPR complex, includingthe guide sequence to be tested and a control guide sequence differentfrom the test guide sequence, and comparing binding or rate of cleavageat the target sequence between the test and control guide sequencereactions. Other assays are possible, and will occur to those skilled inthe art. A guide sequence may be selected to target any target sequence.In some embodiments, the target sequence is a sequence within a genomeof a cell. Exemplary target sequences include those that are unique inthe target genome.

In general, and throughout this specification, the term “vector” refersto a nucleic acid molecule capable of transporting another nucleic acidto which it has been linked. Vectors include, but are not limited to,nucleic acid molecules that are single-stranded, double-stranded, orpartially double-stranded; nucleic acid molecules that comprise one ormore free ends, no free ends (e.g., circular); nucleic acid moleculesthat comprise DNA, RNA, or both; and other varieties of polynucleotidesknown in the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses). Viralvectors also include polynucleotides carried by a virus for transfectioninto a host cell. Certain vectors are capable of autonomous replicationin a host cell into which they are introduced (e.g., bacterial vectorshaving a bacterial origin of replication and episomal mammalianvectors). Other vectors (e.g., non-episomal mammalian vectors) areintegrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively-linked. Such vectors are referred toherein as “expression vectors.” Vectors for and that result inexpression in a eukaryotic cell can be referred to herein as “eukaryoticexpression vectors.” Common expression vectors of utility in recombinantDNA techniques are often in the form of plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell).

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al.,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U 5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. U.S.A., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

Advantageous vectors include lentiviruses and adeno-associated viruses,and types of such vectors can also be selected for targeting particulartypes of cells.

As used herein, the term “crRNA” or “guide RNA” or “single guide RNA” or“sgRNA” or “one or more nucleic acid components” of a Type V or Type VICRISPR-Cas locus effector protein comprises any polynucleotide sequencehaving sufficient complementarity with a target nucleic acid sequence tohybridize with the target nucleic acid sequence and directsequence-specific binding of a nucleic acid-targeting complex to thetarget nucleic acid sequence.

In certain embodiments, the CRISPR system as provided herein can makeuse of a crRNA or analogous polynucleotide comprising a guide sequence,wherein the polynucleotide is an RNA, a DNA or a mixture of RNA and DNA,and/or wherein the polynucleotide comprises one or more nucleotideanalogs. The sequence can comprise any structure, including but notlimited to a structure of a native crRNA, such as a bulge, a hairpin ora stem loop structure. In certain embodiments, the polynucleotidecomprising the guide sequence forms a duplex with a secondpolynucleotide sequence which can be an RNA or a DNA sequence.

In certain embodiments, guides of the invention comprise non-naturallyoccurring nucleic acids and/or non-naturally occurring nucleotidesand/or nucleotide analogs, and/or chemically modifications.Non-naturally occurring nucleic acids can include, for example, mixturesof naturally and non-naturally occurring nucleotides. Non-naturallyoccurring nucleotides and/or nucleotide analogs may be modified at theribose, phosphate, and/or base moiety. In an embodiment of theinvention, a guide nucleic acid comprises ribonucleotides andnon-ribonucleotides. In one such embodiment, a guide comprises one ormore ribonucleotides and one or more deoxyribonucleotides. In anembodiment of the invention, the guide comprises one or morenon-naturally occurring nucleotide or nucleotide analog such as anucleotide with phosphorothioate linkage, boranophosphate linkage, alocked nucleic acid (LNA) nucleotides comprising a methylene bridgebetween the 2′ and 4′ carbons of the ribose ring, or bridged nucleicacids (BNA). Other examples of modified nucleotides include 2′-O-methylanalogs, 2′-deoxy analogs, 2-thiouridine analogs, N6-methyladenosineanalogs, or 2′-fluoro analogs. Further examples of modified basesinclude, but are not limited to, 2-aminopurine, 5-bromo-uridine,pseudouridine (Ψ), N¹-methylpseudouridine (me¹Ψ), 5-methoxyuridine(5moU), inosine, 7-methylguanosine.

In certain embodiments, use is made of chemically modified guide RNAs.Examples of guide RNA chemical modifications include, withoutlimitation, incorporation of 2′-O-methyl (M), 2′-O-methyl3′phosphorothioate (MS), S-constrained ethyl (cEt), or 2′-O-methyl3′thioPACE (MSP) at one or more terminal nucleotides. Such chemicallymodified guide RNAs can comprise increased stability and increasedactivity as compared to unmodified guide RNAs, though on-target vs.off-target specificity is not predictable. (See, Hendel, 2015, NatBiotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, published online 29 Jun.2015; Allerson et al., J. Med. Chem. 2005, 48:901-904; Bramsen et al.,Front. Genet., 2012, 3:154; Deng et al., PNAS, 2015, 112:11870-11875;Sharma et al., Med Chem Comm., 2014, 5:1454-1471; Li et al., NatureBiomedical Engineering, 2017, 1, 0066 DOI:10.1038/s41551-017-0066).Chemically modified guide RNAs further include, without limitation, RNAswith phosphorothioate linkages and locked nucleic acid (LNA) nucleotidescomprising a methylene bridge between the 2′ and 4′ carbons of theribose ring.

In some embodiments, the degree of complementarity, when optimallyaligned using a suitable alignment algorithm, is about or more thanabout 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimalalignment may be determined with the use of any suitable algorithm foraligning sequences, non-limiting example of which include theSmith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithmsbased on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies;available at www.novocraft.com), ELAND (Illumina, San Diego, Calif.),SOAP (available at soap.genomics.org.cn), and Maq (available atmaq.sourceforge.net). The ability of a guide sequence (within a nucleicacid-targeting guide RNA) to direct sequence-specific binding of anucleic acid-targeting complex to a target nucleic acid sequence may beassessed by any suitable assay. For example, the components of a nucleicacid-targeting CRISPR system sufficient to form a nucleic acid-targetingcomplex, including the guide sequence to be tested, may be provided to ahost cell having the corresponding target nucleic acid sequence, such asby transfection with vectors encoding the components of the nucleicacid-targeting complex, followed by an assessment of preferentialtargeting (e.g., cleavage) within the target nucleic acid sequence, suchas by Surveyor assay as described herein. Similarly, cleavage of atarget nucleic acid sequence may be evaluated in a test tube byproviding the target nucleic acid sequence, components of a nucleicacid-targeting complex, including the guide sequence to be tested and acontrol guide sequence different from the test guide sequence, andcomparing binding or rate of cleavage at the target sequence between thetest and control guide sequence reactions. Other assays are possible,and will occur to those skilled in the art. A guide sequence, and hencea nucleic acid-targeting guide RNA may be selected to target any targetnucleic acid sequence. The target sequence may be DNA. The targetsequence may be any RNA sequence. In some embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA),transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA),small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double strandedRNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (lncRNA), andsmall cytoplasmic RNA (scRNA). In some preferred embodiments, the targetsequence may be a sequence within a RNA molecule selected from the groupconsisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments,the target sequence may be a sequence within a RNA molecule selectedfrom the group consisting of ncRNA, and lncRNA. In some more preferredembodiments, the target sequence may be a sequence within an mRNAmolecule or a pre-mRNA molecule.

In some embodiments, a nucleic acid-targeting guide RNA is selected toreduce the degree secondary structure within the RNA-targeting guideRNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%,25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleicacid-targeting guide RNA participate in self-complementary base pairingwhen optimally folded. Optimal folding may be determined by any suitablepolynucleotide folding algorithm. Some programs are based on calculatingthe minimal Gibbs free energy. An example of one such algorithm ismFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981),133-148). Another example folding algorithm is the online webserverRNAfold, developed at Institute for Theoretical Chemistry at theUniversity of Vienna, using the centroid structure prediction algorithm(see e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24; and P A Carrand G M Church, 2009, Nature Biotechnology 27(12): 1151-62).

In certain embodiments, a guide RNA or crRNA may comprise, consistessentially of, or consist of a direct repeat (DR) sequence and a guidesequence or spacer sequence. In certain embodiments, the guide RNA orcrRNA may comprise, consist essentially of, or consist of a directrepeat sequence fused or linked to a guide sequence or spacer sequence.In certain embodiments, the direct repeat sequence may be locatedupstream (i.e., 5′) from the guide sequence or spacer sequence. In otherembodiments, the direct repeat sequence may be located downstream (i.e.,3′) from the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem loop, preferably asingle stem loop. In certain embodiments, the direct repeat sequenceforms a stem loop, preferably a single stem loop.

In certain embodiments, the spacer length of the guide RNA is from 15 to35 nt. In certain embodiments, the spacer length of the guide RNA is atleast 15 nucleotides, preferably at least 18 nt, such that at least 19,20, 21, 22, or more nt. In certain embodiments, the spacer length isfrom 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17,18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26,or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt,e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

Applicants also perform a challenge experiment to verify the RNAtargeting and cleaving capability of a C2c2. This experiment closelyparallels similar work in E. coli for the heterologous expression ofStCas9 (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)).Applicants introduce a plasmid containing both a PAM and a resistancegene into the heterologous E. coli, and then plate on the correspondingantibiotic. If there is RNA cleavage of the plasmid transcribedresistance gene, Applicants observe no viable colonies.

In further detail, the assay is as follows for a DNA target, but may beadapted accordingly for an RNA target. Two E. coli strains are used inthis assay. One carries a plasmid that encodes the endogenous effectorprotein locus from the bacterial strain. The other strain carries anempty plasmid (e.g. pACYC184, control strain). All possible 7 or 8 bpPAM sequences are presented on an antibiotic resistance plasmid (pUC19with ampicillin resistance gene). The PAM is located next to thesequence of proto-spacer 1 (the DNA target to the first spacer in theendogenous effector protein locus). Two PAM libraries were cloned. Onehas a 8 random bp 5′ of the proto-spacer (e.g. total of 65536 differentPAM sequences=complexity). The other library has 7 random bp 3′ of theproto-spacer (e.g. total complexity is 16384 different PAMs). Bothlibraries were cloned to have in average 500 plasmids per possible PAM.Test strain and control strain were transformed with 5′ PAM and 3′ PAMlibrary in separate transformations and transformed cells were platedseparately on ampicillin plates. Recognition and subsequentcutting/interference with the plasmid renders a cell vulnerable toampicillin and prevents growth. Approximately 12 h after transformation,all colonies formed by the test and control strains where harvested andplasmid DNA was isolated. Plasmid DNA was used as template for PCRamplification and subsequent deep sequencing. Representation of all PAMsin the untransformed libraries showed the expected representation ofPAMs in transformed cells. Representation of all PAMs found in controlstrains showed the actual representation. Representation of all PAMs intest strain showed which PAMs are not recognized by the enzyme andcomparison to the control strain allows extracting the sequence of thedepleted PAM.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of nucleic acid-targeting guide RNAdelivered. Optimal concentrations of nucleic acid-targeting guide RNAcan be determined by testing different concentrations in a cellular ornon-human eukaryote animal model and using deep sequencing to analyzethe extent of modification at potential off-target genomic loci. Theconcentration that gives the highest level of on-target modificationwhile minimizing the level of off-target modification should be chosenfor in vivo delivery. The nucleic acid-targeting system is derivedadvantageously from a Type VI CRISPR system. In some embodiments, one ormore elements of a nucleic acid-targeting system is derived from aparticular organism comprising an endogenous RNA-targeting system. Inparticular embodiments, the Type VI RNA-targeting Cas enzyme is C2c2.Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17,Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4,homologues thereof, or modified versions thereof. In embodiments, theType VI protein such as C2c2 as referred to herein also encompasses ahomologue or an orthologue of a Type VI protein such as C2c2. The terms“orthologue” (also referred to as “ortholog” herein) and “homologue”(also referred to as “homolog” herein) are well known in the art. Bymeans of further guidance, a “homologue” of a protein as used herein isa protein of the same species which performs the same or a similarfunction as the protein it is a homologue of Homologous proteins may butneed not be structurally related, or are only partially structurallyrelated. An “orthologue” of a protein as used herein is a protein of adifferent species which performs the same or a similar function as theprotein it is an orthologue of Orthologous proteins may but need not bestructurally related, or are only partially structurally related. Inparticular embodiments, the homologue or orthologue of a Type VI proteinsuch as C2c2 as referred to herein has a sequence homology or identityof at least 80%, more preferably at least 85%, even more preferably atleast 90%, such as for instance at least 95% with a Type VI protein suchas C2c2. In further embodiments, the homologue or orthologue of a TypeVI protein such as C2c2 as referred to herein has a sequence identity ofat least 80%, more preferably at least 85%, even more preferably atleast 90%, such as for instance at least 95% with the wild Type VIprotein such as C2c2.

In an embodiment, the Type VI RNA-targeting Cas protein may be a C2c2ortholog of an organism of a genus which includes but is not limited toLeptotrichia, Listeria, Corynebacterium, Sutterella, Legionella,Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus,Mycoplasma, Bacteroides, Flavivirus, Flavobacterium, Sphaerochaeta,Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum,Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. Species oforganism of such a genus can be as otherwise herein discussed.

Some methods of identifying orthologs of CRISPR-Cas system enzymes mayinvolve identifying tracr sequences in genomes of interest.Identification of tracr sequences may relate to the following steps:Search for the direct repeats or tracr mate sequences in a database toidentify a CRISPR region comprising a CRISPR enzyme. Search forhomologous sequences in the CRISPR region flanking the CRISPR enzyme inboth the sense and antisense directions. Look for transcriptionalterminators and secondary structures. Identify any sequence that is nota direct repeat or a tracr mate sequence but has more than 50% identityto the direct repeat or tracr mate sequence as a potential tracrsequence. Take the potential tracr sequence and analyze fortranscriptional terminator sequences associated therewith.

It will be appreciated that any of the functionalities described hereinmay be engineered into CRISPR enzymes from other orthologs, includingchimeric enzymes comprising fragments from multiple orthologs. Examplesof such orthologs are described elsewhere herein. Thus, chimeric enzymesmay comprise fragments of CRISPR enzyme orthologs of an organism whichincludes but is not limited to Leptotrichia, Listeria, Corynebacterium,Sutterella, Legionella, Treponema, Filifactor, Eubacterium,Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flavivirus,Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter,Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor,Mycoplasma and Campylobacter. A chimeric enzyme can comprise a firstfragment and a second fragment, and the fragments can be of CRISPRenzyme orthologs of organisms of genera herein mentioned or of speciesherein mentioned; advantageously the fragments are from CRISPR enzymeorthologs of different species.

In embodiments, the Type VI RNA-targeting effector protein, inparticular the C2c2 protein as referred to herein also encompasses afunctional variant of C2c2 or a homologue or an orthologue thereof. A“functional variant” of a protein as used herein refers to a variant ofsuch protein which retains at least partially the activity of thatprotein. Functional variants may include mutants (which may beinsertion, deletion, or replacement mutants), including polymorphs, etc.Also included within functional variants are fusion products of suchprotein with another, usually unrelated, nucleic acid, protein,polypeptide or peptide. Functional variants may be naturally occurringor may be man-made. Advantageous embodiments can involve engineered ornon-naturally occurring Type VI RNA-targeting effector protein.

In an embodiment of the invention, there is provided an effector proteinwhich comprises an amino acid sequence having at least 80% sequencehomology to the wild-type sequence of any of Leptotrichia shahii C2c2,Lachnospiraceae bacterium MA2020 C2c2, Lachnospiraceae bacterium NK4A179C2c2, Clostridium aminophilum (DSM 10710) C2c2, Carnobacteriumgallinarum (DSM 4847) C2c2, Paludibacter propionicigenes (WB4) C2c2,Listeria weihenstephanensis (FSL R9-0317) C2c2, Listeriaceae bacterium(FSL M6-0635) C2c2, Listeria newyorkensis (FSL M6-0635) C2c2,Leptotrichia wadei (F0279) C2c2, Rhodobacter capsulatus (SB 1003) C2c2,Rhodobacter capsulatus (R121) C2c2, Rhodobacter capsulatus (DE442) C2c2,Leptotrichia wadei (Lw2) C2c2, or Listeria seeligeri C2c2.

In an embodiment of the invention, the effector protein comprises anamino acid sequence having at least 80% sequence homology to a Type VIeffector protein consensus sequence including but not limited to aconsensus sequence described herein.

In an embodiment of the invention, the effector protein comprises atleast one HEPN domain, including but not limited to HEPN domainsdescribed herein, HEPN domains known in the art, and domains recognizedto be HEPN domains by comparison to consensus sequences and motifs.Several such domains are provided herein. In one non-limiting example, aconsensus sequence can be derived from the sequences of C2c2 orthologsprovided herein.

In an embodiment of the invention, the effector protein comprises one ormore HEPN domains comprising a RxxxxH motif sequence. The RxxxxH motifsequence can be, without limitation, from an HEPN domain describedherein or an HEPN domain known in the art. RxxxxH motifs sequencesfurther include motif sequences created by combining portions of two ormore HEPN domains. As noted, consensus sequences can be derived from thesequences of the 15 orthologs disclosed in U.S. 62/432,240 (BI-10035).For example, from the above sequence alignment, the first HEPN domaincomprises a R{N/H}xxxH motif whereas the second HEPN domain comprises aR{N/K}xxxH motif.

In an embodiment of the invention, a HEPN domain comprises at least oneRxxxxH motif comprising the sequence of R{N/H/K}X₁X₂X₃H. In anembodiment of the invention, a HEPN domain comprises a RxxxxH motifcomprising the sequence of R{N/H}X₁X₂X₃H. In an embodiment of theinvention, a HEPN domain comprises the sequence of R{N/K}X₁X₂X₃H. Incertain embodiments, X₁ is R, S, D, E, Q, N, G, Y, or H. In certainembodiments, X₂ is I, S, T, V, or L. In certain embodiments, X₃ is L, F,N, Y, V, I, S, D, E, or A.

Additional effectors for use according to the invention can beidentified by their proximity to cas1 genes, for example, though notlimited to, within the region 20 kb from the start of the cas1 gene and20 kb from the end of the cas1 gene. In certain embodiments, theeffector protein comprises at least one HEPN domain and at least 500amino acids, and wherein the C2c2 effector protein is naturally presentin a prokaryotic genome within 20 kb upstream or downstream of a Cas1gene or a CRISPR array.

In an embodiment, nucleic acid molecule(s) encoding the Type VIRNA-targeting effector protein, in particular C2c2 or an ortholog orhomolog thereof, may be codon-optimized for expression in an eukaryoticcell. A eukaryote can be as herein discussed. Nucleic acid molecule(s)can be engineered or non-naturally occurring.

In an embodiment, the Type VI RNA-targeting effector protein, inparticular C2c2 or an ortholog or homolog thereof, may comprise one ormore mutations (and hence nucleic acid molecule(s) coding for same mayhave mutation(s)). The mutations may be artificially introducedmutations and may include but are not limited to one or more mutationsin a catalytic domain. Examples of catalytic domains with reference to aCas9 enzyme may include but are not limited to RuvC I, RuvC II, RuvC IIIand HNH domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may comprise one or more mutations. The mutations maybe artificially introduced mutations and may include but are not limitedto one or more mutations in a catalytic domain. Examples of catalyticdomains with reference to a Cas enzyme may include but are not limitedto HEPN domains.

In an embodiment, the Type VI protein such as C2c2 or an ortholog orhomolog thereof, may be used as a generic nucleic acid binding proteinwith fusion to or being operably linked to a functional domain.Exemplary functional domains may include but are not limited totranslational initiator, translational activator, translationalrepressor, nucleases, in particular ribonucleases, a spliceosome, beads,a light inducible/controllable domain or a chemicallyinducible/controllable domain.

In some embodiments, the unmodified nucleic acid-targeting effectorprotein may have cleavage activity. In some embodiments, theRNA-targeting effector protein may direct cleavage of one or bothnucleic acid (DNA or RNA) strands at the location of or near a targetsequence, such as within the target sequence and/or within thecomplement of the target sequence or at sequences associated with thetarget sequence. In some embodiments, the nucleic acid-targeting Casprotein may direct cleavage of one or both DNA or RNA strands withinabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, ormore base pairs from the first or last nucleotide of a target sequence.In some embodiments, a vector encodes a nucleic acid-targeting Casprotein that may be mutated with respect to a corresponding wild-typeenzyme such that the mutated nucleic acid-targeting Cas protein lacksthe ability to cleave RNA strands of a target polynucleotide containinga target sequence. As a further example, two or more catalytic domainsof Cas (e.g. HEPN domain) may be mutated to produce a mutated Cassubstantially lacking all RNA cleavage activity. In some embodiments, anucleic acid-targeting effector protein may be considered tosubstantially lack all RNA cleavage activity when the RNA cleavageactivity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%,0.1%, 0.01%, or less of the nucleic acid cleavage activity of thenon-mutated form of the enzyme; an example can be when the nucleic acidcleavage activity of the mutated form is nil or negligible as comparedwith the non-mutated form. An effector protein may be identified withreference to the general class of enzymes that share homology to thebiggest nuclease with multiple nuclease domains from the Type VI CRISPRsystem. Most preferably, the effector protein is a Type VI protein suchas C2c2. By derived, Applicants mean that the derived enzyme is largelybased, in the sense of having a high degree of sequence homology with, awildtype enzyme, but that it has been mutated (modified) in some way asknown in the art or as described herein.

Again, it will be appreciated that the terms Cas and CRISPR enzyme andCRISPR protein and Cas protein are generally used interchangeably and atall points of reference herein refer by analogy to novel CRISPR effectorproteins further described in this application, unless otherwiseapparent, such as by specific reference to Cas9. As mentioned above,many of the residue numberings used herein refer to the effector proteinfrom the Type VI CRISPR locus. However, it will be appreciated that thisinvention includes many more effector proteins from other species ofmicrobes. In certain embodiments, Cas may be constitutively present orinducibly present or conditionally present or administered or delivered.Cas optimization may be used to enhance function or to develop newfunctions, one can generate chimeric Cas proteins. And Cas may be usedas a generic nucleic acid binding protein.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth DNA or RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 20, 50, 100, 200, 500, or more base pairs from) the targetsequence. As used herein the term “sequence(s) associated with a targetlocus of interest” refers to sequences near the vicinity of the targetsequence (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 200,500, or more base pairs from the target sequence, wherein the targetsequence is comprised within a target locus of interest).

An example of a codon optimized sequence, is in this instance a sequenceoptimized for expression in a eukaryote, e.g., humans (i.e. beingoptimized for expression in humans), or for another eukaryote, animal ormammal as herein discussed; see, e.g., SaCas9 human codon optimizedsequence in WO 2014/093622 (PCT/US2013/074667) as an example of a codonoptimized sequence (from knowledge in the art and this disclosure, codonoptimizing coding nucleic acid molecule(s), especially as to effectorprotein (e.g., C2c2) is within the ambit of the skilled artisan). Whilstthis is preferred, it will be appreciated that other examples arepossible and codon optimization for a host species other than human, orfor codon optimization for specific organs is known. In someembodiments, an enzyme coding sequence encoding a DNA/RNA-targeting Casprotein is codon optimized for expression in particular cells, such aseukaryotic cells. The eukaryotic cells may be those of or derived from aparticular organism, such as a mammal, including but not limited tohuman, or non-human eukaryote or animal or mammal as herein discussed,e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal orprimate. In some embodiments, processes for modifying the germ linegenetic identity of human beings and/or processes for modifying thegenetic identity of animals which are likely to cause them sufferingwithout any substantial medical benefit to man or animal, and alsoanimals resulting from such processes, may be excluded. In general,codon optimization refers to a process of modifying a nucleic acidsequence for enhanced expression in the host cells of interest byreplacing at least one codon (e.g., about or more than about 1, 2, 3, 4,5, 10, 15, 20, 25, 50, or more codons) of the native sequence withcodons that are more frequently or most frequently used in the genes ofthat host cell while maintaining the native amino acid sequence. Variousspecies exhibit particular bias for certain codons of a particular aminoacid. Codon bias (differences in codon usage between organisms) oftencorrelates with the efficiency of translation of messenger RNA (mRNA),which is in turn believed to be dependent on, among other things, theproperties of the codons being translated and the availability ofparticular transfer RNA (tRNA) molecules. The predominance of selectedtRNAs in a cell is generally a reflection of the codons used mostfrequently in peptide synthesis. Accordingly, genes can be tailored foroptimal gene expression in a given organism based on codon optimization.Codon usage tables are readily available, for example, at the “CodonUsage Database” available at www.kazusa.or.jp/codon/ and these tablescan be adapted in a number of ways. See Nakamura, Y., et al. “Codonusage tabulated from the international DNA sequence databases: statusfor the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithmsfor codon optimizing a particular sequence for expression in aparticular host cell are also available, such as Gene Forge (Aptagen;Jacobus, Pa.), are also available. In some embodiments, one or morecodons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons)in a sequence encoding a DNA/RNA-targeting Cas protein corresponds tothe most frequently used codon for a particular amino acid.

In some embodiments, a vector encodes a nucleic acid-targeting effectorprotein such as the C2c2, or an ortholog or homolog thereof comprisingone or more nuclear localization sequences (NLSs) or nuclear exportsignals (NESs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more NLSs or NESs. In some embodiments, the RNA-targetingeffector protein comprises about or more than about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, or more NLSs or NESs at or near the amino-terminus, about ormore than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs or NESs ator near the carboxy-terminus, or a combination of these (e.g., zero orat least one or more NLS or NES at the amino-terminus and zero or at oneor more NLS or NES at the carboxy terminus). When more than one NLS orNES is present, each may be selected independently of the others, suchthat a single NLS or NES may be present in more than one copy and/or incombination with one or more other NLSs or NESs present in one or morecopies. In some embodiments, an NLS or NES is considered near the N- orC-terminus when the nearest amino acid of the NLS or NES is within about1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along thepolypeptide chain from the N- or C-terminus. Non-limiting examples ofNLSs include an NLS sequence derived from: the NLS of the SV40 viruslarge T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 27);the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS withthe sequence KRPAATKKAGQAKKKK (SEQ ID NO: 28)); the c-myc NLS having theamino acid sequence PAAKRVKLD (SEQ ID NO: 29) or RQRRNELKRSP (SEQ ID NO:30); the hRNPA1 M9 NLS having the sequenceNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 31); the sequenceRMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 32) of the IBBdomain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 33) andPPKKARED (SEQ ID NO: 34) of the myoma T protein; the sequence PQPKKKPL(SEQ ID NO: 35) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 36)of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 37) and PKQKKRK (SEQID NO: 38) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ IDNO: 39) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR(SEQ ID NO: 40) of the mouse Mx1 protein; the sequenceKRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 41) of the human poly(ADP-ribose)polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 42) of thesteroid hormone receptors (human) glucocorticoid. In general, the one ormore NLSs or NESs are of sufficient strength to drive accumulation ofthe DNA/RNA-targeting Cas protein in a detectable amount in respectivelythe nucleus or cytoplasm of a eukaryotic cell. In general, strength ofnuclear/cytoplasmic localization activity may derive from the number ofNLSs or NESs in the nucleic acid-targeting effector protein, theparticular NLS(s) or NES(s) used, or a combination of these factors.Detection of accumulation in the nucleus/cytoplasm may be performed byany suitable technique. For example, a detectable marker may be fused tothe nucleic acid-targeting protein, such that location within a cell maybe visualized, such as in combination with a means for detecting thelocation of the nucleus (e.g., a stain specific for the nucleus such asDAPI) or cytoplasm. Cell nuclei may also be isolated from cells, thecontents of which may then be analyzed by any suitable process fordetecting protein, such as immunohistochemistry, Western blot, or enzymeactivity assay. Accumulation in the nucleus/cytoplasm may also bedetermined indirectly, such as by an assay for the effect of nucleicacid-targeting complex formation (e.g., assay for RNA cleavage ormutation at the target sequence, or assay for altered gene expressionactivity affected by RNA-targeting complex formation and/orRNA-targeting Cas protein activity), as compared to a control notexposed to the nucleic acid-targeting Cas protein or nucleicacid-targeting complex, or exposed to a nucleic acid-targeting Casprotein lacking the one or more NLSs or NESs. In preferred embodimentsof the herein described C2c2 effector protein complexes and systems thecodon optimized C2c2 effector proteins comprise an NLS or NES attachedto the C-terminal of the protein.

In some embodiments, one or more vectors driving expression of one ormore elements of a nucleic acid-targeting system are introduced into ahost cell such that expression of the elements of the nucleicacid-targeting system direct formation of a nucleic acid-targetingcomplex at one or more target sites. For example, a nucleicacid-targeting effector enzyme and a nucleic acid-targeting guide RNAcould each be operably linked to separate regulatory elements onseparate vectors. RNA(s) of the nucleic acid-targeting system can bedelivered to a transgenic nucleic acid-targeting effector protein animalor mammal, e.g., an animal or mammal that constitutively or inducibly orconditionally expresses nucleic acid-targeting effector protein; or ananimal or mammal that is otherwise expressing nucleic acid-targetingeffector protein or has cells containing nucleic acid-targeting effectorprotein, such as by way of prior administration thereto of a vector orvectors that code for and express in vivo nucleic acid-targetingeffector protein. Alternatively, two or more of the elements expressedfrom the same or different regulatory elements, may be combined in asingle vector, with one or more additional vectors providing anycomponents of the nucleic acid-targeting system not included in thefirst vector. Nucleic acid-targeting system elements that are combinedin a single vector may be arranged in any suitable orientation, such asone element located 5′ with respect to (“upstream” of) or 3′ withrespect to (“downstream” of) a second element. The coding sequence ofone element may be located on the same or opposite strand of the codingsequence of a second element, and oriented in the same or oppositedirection. In some embodiments, a single promoter drives expression of atranscript encoding a nucleic acid-targeting effector protein and thenucleic acid-targeting guide RNA, embedded within one or more intronsequences (e.g., each in a different intron, two or more in at least oneintron, or all in a single intron). In some embodiments, the nucleicacid-targeting effector protein and the nucleic acid-targeting guide RNAmay be operably linked to and expressed from the same promoter. Deliveryvehicles, vectors, particles, nanoparticles, formulations and componentsthereof for expression of one or more elements of a nucleicacid-targeting system are as used in the foregoing documents, such as WO2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprisesone or more insertion sites, such as a restriction endonucleaserecognition sequence (also referred to as a “cloning site”). In someembodiments, one or more insertion sites (e.g., about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are locatedupstream and/or downstream of one or more sequence elements of one ormore vectors. In some embodiments, a vector comprises two or moreinsertion sites, so as to allow insertion of a guide sequence at eachsite. In such an arrangement, the two or more guide sequences maycomprise two or more copies of a single guide sequence, two or moredifferent guide sequences, or combinations of these. When multipledifferent guide sequences are used, a single expression construct may beused to target nucleic acid-targeting activity to multiple different,corresponding target sequences within a cell. For example, a singlevector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or more guide sequences. In some embodiments, about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more suchguide-sequence-containing vectors may be provided, and optionallydelivered to a cell. In some embodiments, a vector comprises aregulatory element operably linked to an enzyme-coding sequence encodinga nucleic acid-targeting effector protein. Nucleic acid-targetingeffector protein or nucleic acid-targeting guide RNA or RNA(s) can bedelivered separately; and advantageously at least one of these isdelivered via a particle or nanoparticle complex. nucleic acid-targetingeffector protein mRNA can be delivered prior to the nucleicacid-targeting guide RNA to give time for nucleic acid-targetingeffector protein to be expressed. nucleic acid-targeting effectorprotein mRNA might be administered 1-12 hours (preferably around 2-6hours) prior to the administration of nucleic acid-targeting guide RNA.Alternatively, nucleic acid-targeting effector protein mRNA and nucleicacid-targeting guide RNA can be administered together. Advantageously, asecond booster dose of guide RNA can be administered 1-12 hours(preferably around 2-6 hours) after the initial administration ofnucleic acid-targeting effector protein mRNA+guide RNA. Additionaladministrations of nucleic acid-targeting effector protein mRNA and/orguide RNA might be useful to achieve the most efficient levels of genomeand/or transcriptome modification.

In one aspect, the invention provides methods for using one or moreelements of a nucleic acid-targeting system. The nucleic acid-targetingcomplex of the invention provides an effective means for modifying atarget RNA. The nucleic acid-targeting complex of the invention has awide variety of utility including modifying (e.g., deleting, inserting,translocating, inactivating, activating) a target RNA in a multiplicityof cell types. As such the nucleic acid-targeting complex of theinvention has a broad spectrum of applications in, e.g., gene therapy,drug screening, disease diagnosis, and prognosis. An exemplary nucleicacid-targeting complex comprises a RNA-targeting effector proteincomplexed with a guide RNA hybridized to a target sequence within thetarget locus of interest.

In one embodiment, this invention provides a method of cleaving a targetRNA. The method may comprise modifying a target RNA using a nucleicacid-targeting complex that binds to the target RNA and effect cleavageof said target RNA. In an embodiment, the nucleic acid-targeting complexof the invention, when introduced into a cell, may create a break (e.g.,a single or a double strand break) in the RNA sequence. For example, themethod can be used to cleave a disease RNA in a cell. For example, anexogenous RNA template comprising a sequence to be integrated flanked byan upstream sequence and a downstream sequence may be introduced into acell. The upstream and downstream sequences share sequence similaritywith either side of the site of integration in the RNA. Where desired, adonor RNA can be mRNA. The exogenous RNA template comprises a sequenceto be integrated (e.g., a mutated RNA). The sequence for integration maybe a sequence endogenous or exogenous to the cell. Examples of asequence to be integrated include RNA encoding a protein or a non-codingRNA (e.g., a microRNA). Thus, the sequence for integration may beoperably linked to an appropriate control sequence or sequences.Alternatively, the sequence to be integrated may provide a regulatoryfunction. The upstream and downstream sequences in the exogenous RNAtemplate are selected to promote recombination between the RNA sequenceof interest and the donor RNA. The upstream sequence is a RNA sequencethat shares sequence similarity with the RNA sequence upstream of thetargeted site for integration. Similarly, the downstream sequence is aRNA sequence that shares sequence similarity with the RNA sequencedownstream of the targeted site of integration. The upstream anddownstream sequences in the exogenous RNA template can have 75%, 80%,85%, 90%, 95%, or 100% sequence identity with the targeted RNA sequence.Preferably, the upstream and downstream sequences in the exogenous RNAtemplate have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identitywith the targeted RNA sequence. In some methods, the upstream anddownstream sequences in the exogenous RNA template have about 99% or100% sequence identity with the targeted RNA sequence. An upstream ordownstream sequence may comprise from about 20 bp to about 2500 bp, forexample, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200,2300, 2400, or 2500 bp. In some methods, the exemplary upstream ordownstream sequence have about 200 bp to about 2000 bp, about 600 bp toabout 1000 bp, or more particularly about 700 bp to about 1000 bp. Insome methods, the exogenous RNA template may further comprise a marker.Such a marker may make it easy to screen for targeted integrations.Examples of suitable markers include restriction sites, fluorescentproteins, or selectable markers. The exogenous RNA template of theinvention can be constructed using recombinant techniques (see, forexample, Sambrook et al., 2001 and Ausubel et al., 1996). In a methodfor modifying a target RNA by integrating an exogenous RNA template, abreak (e.g., double or single stranded break in double or singlestranded DNA or RNA) is introduced into the DNA or RNA sequence by thenucleic acid-targeting complex, the break is repaired via homologousrecombination with an exogenous RNA template such that the template isintegrated into the RNA target. The presence of a double-stranded breakfacilitates integration of the template. In other embodiments, thisinvention provides a method of modifying expression of a RNA in aeukaryotic cell. The method comprises increasing or decreasingexpression of a target polynucleotide by using a nucleic acid-targetingcomplex that binds to the RNA (e.g., mRNA or pre-mRNA). In some methods,a target RNA can be inactivated to effect the modification of theexpression in a cell. For example, upon the binding of a RNA-targetingcomplex to a target sequence in a cell, the target RNA is inactivatedsuch that the sequence is not translated, the coded protein is notproduced, or the sequence does not function as the wild-type sequencedoes. For example, a protein or microRNA coding sequence may beinactivated such that the protein or microRNA or pre-microRNA transcriptis not produced. The target RNA of a RNA-targeting complex can be anyRNA endogenous or exogenous to the eukaryotic cell. For example, thetarget RNA can be a RNA residing in the nucleus of the eukaryotic cell.The target RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA). Examples of target RNA include a sequence associatedwith a signaling biochemical pathway, e.g., a signaling biochemicalpathway-associated RNA. Examples of target RNA include a diseaseassociated RNA. A “disease-associated” RNA refers to any RNA which isyielding translation products at an abnormal level or in an abnormalform in cells derived from a disease-affected tissues compared withtissues or cells of a non disease control. It may be a RNA transcribedfrom a gene that becomes expressed at an abnormally high level; it maybe a RNA transcribed from a gene that becomes expressed at an abnormallylow level, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated RNA also refersto a RNA transcribed from a gene possessing mutation(s) or geneticvariation that is directly responsible or is in linkage disequilibriumwith a gene(s) that is responsible for the etiology of a disease. Thetranslated products may be known or unknown, and may be at a normal orabnormal level. The target RNA of a RNA-targeting complex can be any RNAendogenous or exogenous to the eukaryotic cell. For example, the targetRNA can be a RNA residing in the nucleus of the eukaryotic cell. Thetarget RNA can be a sequence (e.g., mRNA or pre-mRNA) coding a geneproduct (e.g., a protein) or a non-coding sequence (e.g., ncRNA, lncRNA,tRNA, or rRNA).

In some embodiments, the method may comprise allowing a nucleicacid-targeting complex to bind to the target RNA to effect cleavage ofsaid target RNA or RNA thereby modifying the target RNA, wherein thenucleic acid-targeting complex comprises a nucleic acid-targetingeffector protein complexed with a guide RNA hybridized to a targetsequence within said target RNA. In one aspect, the invention provides amethod of modifying expression of RNA in a eukaryotic cell. In someembodiments, the method comprises allowing a nucleic acid-targetingcomplex to bind to the RNA such that said binding results in increasedor decreased expression of said RNA; wherein the nucleic acid-targetingcomplex comprises a nucleic acid-targeting effector protein complexedwith a guide RNA. Similar considerations and conditions apply as abovefor methods of modifying a target RNA. In fact, these sampling,culturing and re-introduction options apply across the aspects of thepresent invention. In one aspect, the invention provides for methods ofmodifying a target RNA in a eukaryotic cell, which may be in vivo, exvivo or in vitro. In some embodiments, the method comprises sampling acell or population of cells from a human or non-human animal, andmodifying the cell or cells. Culturing may occur at any stage ex vivo.The cell or cells may even be re-introduced into the non-human animal orplant. For re-introduced cells it is particularly preferred that thecells are stem cells.

Indeed, in any aspect of the invention, the nucleic acid-targetingcomplex may comprise a nucleic acid-targeting effector protein complexedwith a guide RNA hybridized to a target sequence.

The invention relates to the engineering and optimization of systems,methods and compositions used for the control of gene expressioninvolving RNA sequence targeting, that relate to the nucleicacid-targeting system and components thereof. In advantageousembodiments, the effector protein enzyme is a Type VI protein such asC2c2. An advantage of the present methods is that the CRISPR systemminimizes or avoids off-target binding and its resulting side effects.This is achieved using systems arranged to have a high degree ofsequence specificity for the target RNA.

In relation to a nucleic acid-targeting complex or system preferably,the tracr sequence has one or more hairpins and is 30 or morenucleotides in length, 40 or more nucleotides in length, or 50 or morenucleotides in length; the crRNA sequence is between 10 to 30nucleotides in length, the nucleic acid-targeting effector protein is aType VI effector protein.

In certain embodiments, the effector protein may be a Listeria sp.C2c2p, preferably Listeria seeligeri C2c2p, more preferably Listeriaseeligeri serovar 1/2b str. SLCC3954 C2c2p and the crRNA sequence may be44 to 47 nucleotides in length, with a 5′ 29-nt direct repeat (DR) and a15-nt to 18-nt spacer.

In certain embodiments, the effector protein may be a Leptotrichia sp.C2c2p, preferably Leptotrichia shahii C2c2p, more preferablyLeptotrichia shahii DSM 19757 C2c2p and the crRNA sequence may be 42 to58 nucleotides in length, with a 5′direct repeat of at least 24 nt, suchas a 5′ 24-28-nt direct repeat (DR) and a spacer of at least 14 nt, suchas a 14-nt to 28-nt spacer, or a spacer of at least 18 nt, such as 19,20, 21, 22, or more nt, such as 18-28, 19-28, 20-28, 21-28, or 22-28 nt.

More preferably, the effector protein may be a Leptotrichia sp.,preferably Leptotrichia wadei F0279, or a Listeria sp., preferablyListeria newyorkensis FSL M6-0635.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the crRNA sequence maybe 36 to 63 nucleotides in length, preferably 37-nt to 62-nt in length,or 38-nt to 61-nt in length, or 39-nt to 60-nt in length, morepreferably 40-nt to 59-nt in length, or 41-nt to 58-nt in length, mostpreferably 42-nt to 57-nt in length. For example, the crRNA maycomprise, consist essentially of or consist of a direct repeat (DR),preferably a 5′ DR, 26-nt to 31-nt in length, preferably 27-nt to 30-ntin length, even more preferably 28-nt or 29-nt in length or at least 28or 29 nt in length, and a spacer 10-nt to 32-nt in length, preferably11-nt to 31-nt in length, more preferably 12-nt to 30-nt in length, evenmore preferably 13-nt to 29-nt in length, and most preferably 14-nt to28-nt in length, such as 18-28 nt, 19-28 nt, 20-28 nt, 21-28 nt, or22-28 nt.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and the tracrRNA sequence(if present) may be at least 60-nt long, such as at least 65-nt inlength, or at least 70-nt in length, such as from 60-nt to 70-nt inlength, or from 60-nt to 70-nt in length, or from 70-nt to 80-nt inlength, or from 80-nt to 90-nt in length, or from 90-nt to 100-nt inlength, or from 100-nt to 110-nt in length, or from 110-nt to 120-nt inlength, or from 120-nt to 130-nt in length, or from 130-nt to 140-nt inlength, or from 140-nt to 150-nt in length, or more than 150-nt inlength.

In certain embodiments, the effector protein may be a Type VI locieffector protein, more particularly a C2c2p, and no tracrRNA may berequired for cleavage.

The use of two different aptamers (each associated with a distinctnucleic acid-targeting guide RNAs) allows an activator-adaptor proteinfusion and a repressor-adaptor protein fusion to be used, with differentnucleic acid-targeting guide RNAs, to activate expression of one DNA orRNA, whilst repressing another. They, along with their different guideRNAs can be administered together, or substantially together, in amultiplexed approach. A large number of such modified nucleicacid-targeting guide RNAs can be used all at the same time, for example10 or 20 or 30 and so forth, whilst only one (or at least a minimalnumber) of effector protein molecules need to be delivered, as acomparatively small number of effector protein molecules can be usedwith a large number of modified guides. The adaptor protein may beassociated (preferably linked or fused to) one or more activators or oneor more repressors. For example, the adaptor protein may be associatedwith a first activator and a second activator. The first and secondactivators may be the same, but they are preferably differentactivators. Three or more or even four or more activators (orrepressors) may be used, but package size may limit the number beinghigher than 5 different functional domains. Linkers are preferably used,over a direct fusion to the adaptor protein, where two or morefunctional domains are associated with the adaptor protein. Suitablelinkers might include the Gly-Ser linker.

It is also envisaged that the nucleic acid-targeting effectorprotein-guide RNA complex as a whole may be associated with two or morefunctional domains. For example, there may be two or more functionaldomains associated with the nucleic acid-targeting effector protein, orthere may be two or more functional domains associated with the guideRNA (via one or more adaptor proteins), or there may be one or morefunctional domains associated with the nucleic acid-targeting effectorprotein and one or more functional domains associated with the guide RNA(via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressormay include a linker. For example, Gly-Ser linkers GGGS (SEQ ID NO: 48)can be used. They can be used in repeats of 3 ((GGGGS)₃ (SEQ ID NO: 47))or 6 (SEQ ID NO: 49), 9 (SEQ ID NO: 50) or even 12 (SEQ ID NO: 51) ormore, to provide suitable lengths, as required. Linkers can be usedbetween the guide RNAs and the functional domain (activator orrepressor), or between the nucleic acid-targeting effector protein andthe functional domain (activator or repressor). The linkers the user toengineer appropriate amounts of “mechanical flexibility”.

The invention comprehends a nucleic acid-targeting complex comprising anucleic acid-targeting effector protein and a guide RNA, wherein thenucleic acid-targeting effector protein comprises at least one mutation,such that the nucleic acid-targeting Cas protein has no more than 5% ofthe activity of the nucleic acid-targeting Cas protein not having the atleast one mutation and, optionally, at least one or more nuclearlocalization sequences; the guide RNA comprises a guide sequence capableof hybridizing to a target sequence in a RNA of interest in a cell; andwherein: the nucleic acid-targeting effector protein is associated withtwo or more functional domains; or at least one loop of the guide RNA ismodified by the insertion of distinct RNA sequence(s) that bind to oneor more adaptor proteins, and wherein the adaptor protein is associatedwith two or more functional domains; or the nucleic acid-targetingeffector protein is associated with one or more functional domains andat least one loop of the guide RNA is modified by the insertion ofdistinct RNA sequence(s) that bind to one or more adaptor proteins, andwherein the adaptor protein is associated with one or more functionaldomains.

Delivery Generally

C2c2 Effector Protein Complexes can Deliver Functional Effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently eliminatesexpression by mutating the gene at the DNA level, CRISPR-Cas knockdownallows for temporary reduction of gene expression through the use ofartificial transcription or translation factors. Mutating key residuesin both DNA or RNA cleavage domains of the C2c2 protein results in thegeneration of a catalytically inactive C2c2. A catalytically inactiveC2c2 complexes with a guide RNA and localizes to the RNA sequencespecified by that guide RNA's targeting domain, however, it does notcleave the target RNA. Fusion of the inactive C2c2 protein to aneffector domain, e.g., a transcription or translation repression domain,enables recruitment of the effector to any RNA site specified by theguide RNA. In certain embodiments, C2c2 may be fused to atranscriptional repression domain and recruited to the promoter regionof a gene. Especially for gene repression, it is contemplated hereinthat blocking the binding site of an endogenous transcription factorwould aid in downregulating gene expression. In another embodiment, aninactive C2c2 can be fused to a chromatin modifying protein. Alteringchromatin status can result in decreased expression of the target gene.In further embodiments, C2c2 may be fused to a translation repressiondomain.

In an embodiment, a guide RNA molecule can be targeted to a knowntranscription response elements (e.g., promoters, enhancers, etc.), aknown upstream activating sequence, and/or sequences of unknown or knownfunction that are suspected of being able to control (protein)expression of the target RNA.

In some methods, a target polynucleotide can be inactivated to effectthe modification of the expression in a cell. For example, upon thebinding of a CRISPR complex to a target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not transcribed,the coded protein is not produced, or the sequence does not function asthe wild-type sequence does. For example, a protein or microRNA codingsequence may be inactivated such that the protein is not produced. Insome methods, a target polynucleotide can be inactivated to effect themodification of the expression in a cell. For example, upon the bindingof a CRISPR complex to an RNA target sequence in a cell, the targetpolynucleotide is inactivated such that the sequence is not translated,affecting the expression level of the protein in the cell.

In particular embodiments, the CRISPR enzyme comprises one or moremutations selected from the group consisting of R597A, H602A, R1278A andH1283A and/or the one or more mutations are in the HEPN domain of theCRISPR enzyme or is a mutation as otherwise discussed herein. In someembodiments, the CRISPR enzyme has one or more mutations in a catalyticdomain, wherein when transcribed, the direct repeat sequence forms asingle stem loop and the guide sequence directs sequence-specificbinding of a CRISPR complex to the target sequence, and wherein theenzyme further comprises a functional domain. In some embodiments, thefunctional domain is a. In some embodiments, the functional domain is atranscription repression domain, preferably KRAB. In some embodiments,the transcription repression domain is SID, or concatemers of SID (e.g.SID4X). In some embodiments, the functional domain is an epigeneticmodifying domain, such that an epigenetic modifying enzyme is provided.In some embodiments, the functional domain is an activation domain,which may be the P65 activation domain.

Delivery of the C2c2 Effector Protein Complex or Components Thereof

Through this disclosure and the knowledge in the art, TALEs, CRISPR-Cassystems, or components thereof or nucleic acid molecules thereof ornucleic acid molecules encoding or providing components thereof may bedelivered by a delivery system herein described both generally and indetail.

Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, forinstance a Type V protein such as C2c2, and/or any of the present RNAs,for instance a guide RNA, can be delivered using any suitable vector,e.g., plasmid or viral vectors, such as adeno associated virus (AAV),lentivirus, adenovirus or other viral vector types, or combinationsthereof. Effector proteins and one or more guide RNAs can be packagedinto one or more vectors, e.g., plasmid or viral vectors. In someembodiments, the vector, e.g., plasmid or viral vector is delivered tothe tissue of interest by, for example, an intramuscular injection,while other times the delivery is via intravenous, transdermal,intranasal, oral, mucosal, or other delivery methods. Such delivery maybe either via a single dose, or multiple doses. One skilled in the artunderstands that the actual dosage to be delivered herein may varygreatly depending upon a variety of factors, such as the vector choice,the target cell, organism, or tissue, the general condition of thesubject to be treated, the degree of transformation/modification sought,the administration route, the administration mode, the type oftransformation/modification sought, etc.

Such a dosage may further contain, for example, a carrier (water,saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, apharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), apharmaceutically-acceptable excipient, and/or other compounds known inthe art. The dosage may further contain one or more pharmaceuticallyacceptable salts such as, for example, a mineral acid salt such as ahydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and thesalts of organic acids such as acetates, propionates, malonates,benzoates, etc. Additionally, auxiliary substances, such as wetting oremulsifying agents, pH buffering substances, gels or gelling materials,flavorings, colorants, microspheres, polymers, suspension agents, etc.may also be present herein. In addition, one or more other conventionalpharmaceutical ingredients, such as preservatives, humectants,suspending agents, surfactants, antioxidants, anticaking agents,fillers, chelating agents, coating agents, chemical stabilizers, etc.may also be present, especially if the dosage form is a reconstitutableform. Suitable exemplary ingredients include microcrystalline cellulose,carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol,chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propylgallate, the parabens, ethyl vanillin, glycerin, phenol,parachlorophenol, gelatin, albumin and a combination thereof. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which isincorporated by reference herein.

In an embodiment herein the delivery is via an adenovirus, which may beat a single booster dose containing at least 1×10⁵ particles (alsoreferred to as particle units, pu) of adenoviral vector. In anembodiment herein, the dose preferably is at least about 1×10⁶ particles(for example, about 1×10⁶-1×10¹² particles), more preferably at leastabout 1×10⁷ particles, more preferably at least about 1×10⁸ particles(e.g., about 1×10⁸-1×10¹¹ particles or about 1×10⁸-1×10¹² particles),and most preferably at least about 1×10⁹ particles (e.g., about1×10⁹-1×10¹⁰ particles or about 1×10⁹-1×10¹² particles), or even atleast about 1×10¹⁰ particles (e.g., about 1×10¹⁰-1×10¹² particles) ofthe adenoviral vector. Alternatively, the dose comprises no more thanabout 1×10¹⁴ particles, preferably no more than about 1×10¹³ particles,even more preferably no more than about 1×10¹² particles, even morepreferably no more than about 1×10¹¹ particles, and most preferably nomore than about 1×10¹⁰ particles (e.g., no more than about 1×10⁹articles). Thus, the dose may contain a single dose of adenoviral vectorwith, for example, about 1×10⁶ particle units (pu), about 2×10⁶ pu,about 4×10⁶ pu, about 1×10⁷ pu, about 2×10⁷ pu, about 4×10⁷ pu, about1×10⁸ pu, about 2×10⁸ pu, about 4×10⁸ pu, about 1×10⁹ pu, about 2×10⁹pu, about 4×10⁹ pu, about 1×10¹⁰ pu, about 2×10¹⁰ pu, about 4×10¹⁰ pu,about 1×10¹¹ pu, about 2×10¹¹ pu, about 4×10¹¹ pu, about 1×10¹² pu,about 2×10¹² pu, or about 4×10¹² pu of adenoviral vector. See, forexample, the adenoviral vectors in U.S. Pat. No. 8,454,972 B2 to Nabel,et al., granted on Jun. 4, 2013; incorporated by reference herein, andthe dosages at col 29, lines 36-58 thereof. In an embodiment herein, theadenovirus is delivered via multiple doses.

In an embodiment herein, the delivery is via an AAV. A therapeuticallyeffective dosage for in vivo delivery of the AAV to a human is believedto be in the range of from about 20 to about 50 ml of saline solutioncontaining from about 1×10¹⁰ to about 1×10¹⁰ functional AAV/ml solution.The dosage may be adjusted to balance the therapeutic benefit againstany side effects. In an embodiment herein, the AAV dose is generally inthe range of concentrations of from about 1×10⁵ to 1×10⁵⁰ genomes AAV,from about 1×10⁸ to 1×10²⁰ genomes AAV, from about 1×10¹⁰ to about1×10¹⁶ genomes, or about 1×10¹¹ to about 1×10¹⁶ genomes AAV. A humandosage may be about 1×10¹³ genomes AAV. Such concentrations may bedelivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50ml, or about 10 to about 25 ml of a carrier solution. Other effectivedosages can be readily established by one of ordinary skill in the artthrough routine trials establishing dose response curves. See, forexample, U.S. Pat. No. 8,404,658 B2 to Hajjar, et al., granted on Mar.26, 2013, at col. 27, lines 45-60.

In an embodiment herein the delivery is via a plasmid. In such plasmidcompositions, the dosage should be a sufficient amount of plasmid toelicit a response. For instance, suitable quantities of plasmid DNA inplasmid compositions can be from about 0.1 to about 2 mg, or from about1 μg to about 10 μg per 70 kg individual. Plasmids of the invention willgenerally comprise (i) a promoter; (ii) a sequence encoding an nucleicacid-targeting CRISPR enzyme, operably linked to said promoter; (iii) aselectable marker; (iv) an origin of replication; and (v) atranscription terminator downstream of and operably linked to (ii). Theplasmid can also encode the RNA components of a CRISPR complex, but oneor more of these may instead be encoded on a different vector.

The doses herein are based on an average 70 kg individual. The frequencyof administration is within the ambit of the medical or veterinarypractitioner (e.g., physician, veterinarian), or scientist skilled inthe art. It is also noted that mice used in experiments are typicallyabout 20 g and from mice experiments one can scale up to a 70 kgindividual.

In some embodiments the RNA molecules of the invention are delivered inliposome or lipofectin formulations and the like and can be prepared bymethods well known to those skilled in the art. Such methods aredescribed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and5,580,859, which are herein incorporated by reference. Delivery systemsaimed specifically at the enhanced and improved delivery of siRNA intomammalian cells have been developed, (see, for example, Shen et al. FEBSLet. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010;Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol.Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 andSimeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to thepresent invention. siRNA has recently been successfully used forinhibition of gene expression in primates (see for example, Tolentino etal., Retina 24(4):660) which may also be applied to the presentinvention.

Indeed, RNA delivery is a useful method of in vivo delivery. It ispossible to deliver nucleic acid-targeting Cas protein Cas9 and guideRNA gRNA (and, for instance, HR repair template) into cells usingliposomes or particles. Thus delivery of the nucleic acid-targeting Casprotein/CRISPR enzyme, such as a Cas9 and/or delivery of the guide RNAsof the invention may be in RNA form and via microvesicles, liposomes orparticles. For example, Cas mRNA and guide RNA can be packaged intoliposomal particles for delivery in vivo. Liposomal transfectionreagents such as lipofectamine from Life Technologies and other reagentson the market can effectively deliver RNA molecules into the liver.

Means of delivery of RNA also preferred include delivery of RNA viananoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei,Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticlesfor small interfering RNA delivery to endothelial cells, AdvancedFunctional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A.,Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-basednanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267:9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to beparticularly useful in delivery siRNA, a system with some parallels tothe RNA-targeting system. For instance, El-Andaloussi S, et al.(“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc.2012 December; 7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012Nov. 15.) describe how exosomes are promising tools for drug deliveryacross different biological barriers and can be harnessed for deliveryof siRNA in vitro and in vivo. Their approach is to generate targetedexosomes through transfection of an expression vector, comprising anexosomal protein fused with a peptide ligand. The exosomes are thenpurify and characterized from transfected cell supernatant, then RNA isloaded into the exosomes. Delivery or administration according to theinvention can be performed with exosomes, in particular but not limitedto the brain. Vitamin E (a-tocopherol) may be conjugated with nucleicacid-targeting Cas protein and delivered to the brain along with highdensity lipoprotein (HDL), for example in a similar manner as was doneby Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for deliveringshort-interfering RNA (siRNA) to the brain. Mice were infused viaOsmotic minipumps (model 1007D; Alzet, Cupertino, Calif.) filled withphosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL andconnected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannulawas placed about 0.5 mm posterior to the bregma at midline for infusioninto the dorsal third ventricle. Uno et al. found that as little as 3nmol of Toc-siRNA with HDL could induce a target reduction in comparabledegree by the same ICV infusion method. A similar dosage of nucleicacid-targeting effector protein conjugated to a-tocopherol andco-administered with HDL targeted to the brain may be contemplated forhumans in the present invention, for example, about 3 nmol to about 3μmol of nucleic acid-targeting effector protein targeted to the brainmay be contemplated. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April2011)) describes a method of lentiviral-mediated delivery ofshort-hairpin RNAs targeting PKCγ for in vivo gene silencing in thespinal cord of rats. Zou et al. administered about 10 μl of arecombinant lentivirus having a titer of 1×10⁹ transducing units (TU)/mlby an intrathecal catheter. A similar dosage of nucleic acid-targetingeffector protein expressed in a lentiviral vector targeted to the brainmay be contemplated for humans in the present invention, for example,about 10-50 ml of nucleic acid-targeting effector protein targeted tothe brain in a lentivirus having a titer of 1×10⁹ transducing units(TU)/ml may be contemplated.

In terms of local delivery to the brain, this can be achieved in variousways. For instance, material can be delivered intrastriatally e.g., byinjection. Injection can be performed stereotactically via a craniotomy.

Packaging and Promoters Generally

Ways to package nucleic acid-targeting effector coding nucleic acidmolecules, e.g., DNA, into vectors, e.g., viral vectors, to mediategenome modification in vivo include:

To achieve NHEJ-mediated gene knockout:

Single Virus Vector:

Vector containing two or more expression cassettes:

Promoter—nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Promoter—guide RNA1-terminator

Promoter—guide RNA (N)-terminator (up to size limit of vector)

Double Virus Vector:

Vector 1 containing one expression cassette for driving the expressionof nucleic acid-targeting effector protein

Promoter—nucleic acid-targeting effector protein coding nucleic acidmolecule-terminator

Vector 2 containing one more expression cassettes for driving theexpression of one or more guide RNAs

Promoter—guide RNA1-terminator

Promoter—guide RNA1 (N)-terminator (up to size limit of vector)

To mediate homology-directed repair.

-   -   In addition to the single and double virus vector approaches        described above, an additional vector is used to deliver a        homology-direct repair template.

The promoter used to drive nucleic acid-targeting effector proteincoding nucleic acid molecule expression can include:

AAV ITR can serve as a promoter: this is advantageous for eliminatingthe need for an additional promoter element (which can take up space inthe vector). The additional space freed up can be used to drive theexpression of additional elements (gRNA, etc.). Also, ITR activity isrelatively weaker, so can be used to reduce potential toxicity due toover expression of nucleic acid-targeting effector protein.

For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40,Ferritin heavy or light chains, etc.

For brain or other CNS expression, can use promoters: Synapsin I for allneurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT forGABAergic neurons, etc.

For liver expression, can use Albumin promoter.

For lung expression, can use SP-B.

For endothelial cells, can use ICAM.

For hematopoietic cells can use IFNbeta or CD45.

For Osteoblasts can use OG-2.

The promoter used to drive guide RNA can include:

Pol III promoters such as U6 or H1

Use of Pol II promoter and intronic cassettes to express guide RNA

Adeno Associated Virus (AAV)

Nucleic acid-targeting effector protein and one or more guide RNA can bedelivered using adeno associated virus (AAV), lentivirus, adenovirus orother plasmid or viral vector types, in particular, using formulationsand doses from, for example, U.S. Pat. No. 8,454,972 (formulations,doses for adenovirus), U.S. Pat. No. 8,404,658 (formulations, doses forAAV) and U.S. Pat. No. 5,846,946 (formulations, doses for DNA plasmids)and from clinical trials and publications regarding the clinical trialsinvolving lentivirus, AAV and adenovirus. For examples, for AAV, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,454,972 and as in clinical trials involving AAV. For Adenovirus, theroute of administration, formulation and dose can be as in U.S. Pat. No.8,404,658 and as in clinical trials involving adenovirus. For plasmiddelivery, the route of administration, formulation and dose can be as inU.S. Pat. No. 5,846,946 and as in clinical studies involving plasmids.Doses may be based on or extrapolated to an average 70 kg individual(e.g., a male adult human), and can be adjusted for patients, subjects,mammals of different weight and species. Frequency of administration iswithin the ambit of the medical or veterinary practitioner (e.g.,physician, veterinarian), depending on usual factors including the age,sex, general health, other conditions of the patient or subject and theparticular condition or symptoms being addressed. The viral vectors canbe injected into the tissue of interest. For cell-type specificgenome/transcriptome modification, the expression of nucleicacid-targeting effector protein can be driven by a cell-type specificpromoter. For example, liver-specific expression might use the Albuminpromoter and neuron-specific expression (e.g., for targeting CNSdisorders) might use the Synapsin I promoter.

In terms of in vivo delivery, AAV is advantageous over other viralvectors for a couple of reasons:

-   -   Low toxicity (this may be due to the purification method not        requiring ultracentrifugation of cell particles that can        activate the immune response) and    -   Low probability of causing insertional mutagenesis because it        doesn't integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 Kb. This means that nucleicacid-targeting effector protein (such as a Type V protein such as C2c2)as well as a promoter and transcription terminator have to be all fitinto the same viral vector. Therefore embodiments of the inventioninclude utilizing homologs of nucleic acid-targeting effector proteinthat are shorter.

As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof.One can select the AAV of the AAV with regard to the cells to betargeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsidAAV1, AAV2, AAV5 or any combination thereof for targeting brain orneuronal cells; and one can select AAV4 for targeting cardiac tissue.AAV8 is useful for delivery to the liver. The herein promoters andvectors are preferred individually. A tabulation of certain AAVserotypes as to these cells (see Grimm, D. et al., J. Virol. 82:5887-5911 (2008)) is as follows:

Cell Line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 1002.5 0.0 0.1 10 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 1002.0 0.1 6.7 1 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND Hep1A 20 100 0.21.0 0.1 1 0.2 0.0 911 17 100 11 0.2 0.1 17 0.1 ND CHO 100 100 14 1.4 33350 10    1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 101.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 ND A549 14 100 20 ND 0.5 10 0.50.1 HT1180 20 100 10 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 1251429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222 100ND ND 333 3333 ND NDLentivirus

Lentiviruses are complex retroviruses that have the ability to infectand express their genes in both mitotic and post-mitotic cells. The mostcommonly known lentivirus is the human immunodeficiency virus (HIV),which uses the envelope glycoproteins of other viruses to target a broadrange of cell types.

Lentiviruses may be prepared as follows. After cloning pCas ES10 (whichcontains a lentiviral transfer plasmid backbone), HEK293FT at lowpassage (p=5) were seeded in a T-75 flask to 50% confluence the daybefore transfection in DMEM with 10% fetal bovine serum and withoutantibiotics. After 20 hours, media was changed to Opti-MEM (serum-free)media and transfection was done 4 hours later. Cells were transfectedwith 10 μg of lentiviral transfer plasmid (pCas ES10) and the followingpackaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype), and 7.5 μg ofpsPAX2 (gag/pol/rev/tat). Transfection was done in 4 mL Opti-MEM with acationic lipid delivery agent (50 μL Lipofectamine 2000 and 100 μL Plusreagent). After 6 hours, the media was changed to antibiotic-free DMEMwith 10% fetal bovine serum. These methods use serum during cellculture, but serum-free methods are preferred.

Lentivirus may be purified as follows. Viral supernatants were harvestedafter 48 hours. Supernatants were first cleared of debris and filteredthrough a 0.45 μm low protein binding (PVDF) filter. They were then spunin a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets wereresuspended in 50 μl of DMEM overnight at 4 C. They were then aliquotedand immediately frozen at −80° C.

In another embodiment, minimal non-primate lentiviral vectors based onthe equine infectious anemia virus (EIAV) are also contemplated,especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med2006; 8: 275-285). In another embodiment, RetinoStat®, an equineinfectious anemia virus-based lentiviral gene therapy vector thatexpresses angiostatic proteins endostatin and angiostatin that isdelivered via a subretinal injection for the treatment of the web formof age-related macular degeneration is also contemplated (see, e.g.,Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and thisvector may be modified for the nucleic acid-targeting system of thepresent invention.

In another embodiment, self-inactivating lentiviral vectors with ansiRNA targeting a common exon shared by HIV tat/rev, anucleolar-localizing TAR decoy, and an anti-CCR5-specific hammerheadribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) maybe used and/or adapted to the nucleic acid-targeting system of thepresent invention. A minimum of 2.5×10⁶ CD34+ cells per kilogram patientweight may be collected and prestimulated for 16 to 20 hours in X-VIVO15 medium (Lonza) containing 2 μmol/L-glutamine, stem cell factor (100ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml)(CellGenix) at a density of 2×10⁶ cells/ml. Prestimulated cells may betransduced with lentiviral at a multiplicity of infection of 5 for 16 to24 hours in 75-cm² tissue culture flasks coated with fibronectin (25mg/cm²) (RetroNectin, Takara Bio Inc.).

Lentiviral vectors have been disclosed as in the treatment forParkinson's Disease, see, e.g., US Patent Publication No. 20120295960and U.S. Pat. Nos. 7,303,910 and 7,351,585. Lentiviral vectors have alsobeen disclosed for the treatment of ocular diseases, see e.g., US PatentPublication Nos. 20060281180, 20090007284, US20110117189; US20090017543;US20070054961, US20100317109. Lentiviral vectors have also beendisclosed for delivery to the brain, see, e.g., US Patent PublicationNos. US20110293571, US20040013648, US20070025970, US20090111106 and U.S.Pat. No. 7,259,015.

RNA Delivery

RNA delivery: The nucleic acid-targeting Cas protein, for instance aType V protein such as C2c2, and/or guide RNA, can also be delivered inthe form of RNA. Nucleic acid-targeting Cas protein (such as a Type VIprotein such as C2c2) mRNA can be generated using in vitrotranscription. For example, nucleic acid-targeting effector protein(such as a Type V protein such as C2c2) mRNA can be synthesized using aPCR cassette containing the following elements: T7_promoter-kozaksequence (GCCACC)-effector protein-3′ UTR from beta globin-polyA tail (astring of 120 or more adenines). The cassette can be used fortranscription by T7 polymerase. Guide RNAs can also be transcribed usingin vitro transcription from a cassette containing T7_promoter-GG-guideRNA sequence.

To enhance expression and reduce possible toxicity, the nucleicacid-targeting effector protein-coding sequence and/or the guide RNA canbe modified to include one or more modified nucleoside e.g., usingpseudo-U or 5-Methyl-C.

mRNA delivery methods are especially promising for liver deliverycurrently.

Much clinical work on RNA delivery has focused on RNAi or antisense, butthese systems can be adapted for delivery of RNA for implementing thepresent invention. References below to RNAi etc. should be readaccordingly.

Particle Delivery Systems and/or Formulations:

Several types of particle delivery systems and/or formulations are knownto be useful in a diverse spectrum of biomedical applications. Ingeneral, a particle is defined as a small object that behaves as a wholeunit with respect to its transport and properties. Particles are furtherclassified according to diameter. Coarse particles cover a range between2,500 and 10,000 nanometers. Fine particles are sized between 100 and2,500 nanometers. Ultrafine particles, or nanoparticles, are generallybetween 1 and 100 nanometers in size. The basis of the 100-nm limit isthe fact that novel properties that differentiate particles from thebulk material typically develop at a critical length scale of under 100nm.

As used herein, a particle delivery system/formulation is defined as anybiological delivery system/formulation which includes a particle inaccordance with the present invention. A particle in accordance with thepresent invention is any entity having a greatest dimension (e.g.diameter) of less than 100 microns (μm). In some embodiments, inventiveparticles have a greatest dimension of less than 10 μm. In someembodiments, inventive particles have a greatest dimension of less than2000 nanometers (nm). In some embodiments, inventive particles have agreatest dimension of less than 1000 nanometers (nm). In someembodiments, inventive particles have a greatest dimension of less than900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100nm. Typically, inventive particles have a greatest dimension (e.g.,diameter) of 500 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 250 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 200 nm or less. In some embodiments, inventive particleshave a greatest dimension (e.g., diameter) of 150 nm or less. In someembodiments, inventive particles have a greatest dimension (e.g.,diameter) of 100 nm or less. Smaller particles, e.g., having a greatestdimension of 50 nm or less are used in some embodiments of theinvention. In some embodiments, inventive particles have a greatestdimension ranging between 25 nm and 200 nm.

Particle characterization (including e.g., characterizing morphology,dimension, etc.) is done using a variety of different techniques. Commontechniques are electron microscopy (TEM, SEM), atomic force microscopy(AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy(XPS), powder X-ray diffraction (XRD), Fourier transform infraredspectroscopy (FTIR), matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visiblespectroscopy, dual polarisation interferometry and nuclear magneticresonance (NMR). Characterization (dimension measurements) may be madeas to native particles (i.e., preloading) or after loading of the cargo(herein cargo refers to e.g., one or more components of CRISPR-Cassystem e.g., CRISPR enzyme or mRNA or guide RNA, or any combinationthereof, and may include additional carriers and/or excipients) toprovide particles of an optimal size for delivery for any in vitro, exvivo and/or in vivo application of the present invention. In certainpreferred embodiments, particle dimension (e.g., diameter)characterization is based on measurements using dynamic laser scattering(DLS). Mention is made of U.S. Pat. Nos. 8,709,843; 6,007,845;5,855,913; 5,985,309; 5,543,158; and the publication by James E. Dahlmanand Carmen Barnes et al. Nature Nanotechnology (2014) published online11 May 2014, doi:10.1038/nnano.2014.84, concerning particles, methods ofmaking and using them and measurements thereof.

Particles delivery systems within the scope of the present invention maybe provided in any form, including but not limited to solid, semi-solid,emulsion, or colloidal particles. As such any of the delivery systemsdescribed herein, including but not limited to, e.g., lipid-basedsystems, liposomes, micelles, microvesicles, exosomes, or gene gun maybe provided as particle delivery systems within the scope of the presentinvention.

Particles

CRISPR enzyme mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes; for instance, CRISPR enzyme and RNA of theinvention, e.g., as a complex, can be delivered via a particle as inDahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1(see, e.g., James E. Dahlman and Carmen Barnes et al. NatureNanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84), e.g., delivery particle comprising lipid orlipidoid and hydrophilic polymer, e.g., cationic lipid and hydrophilicpolymer, for instance wherein the the cationic lipid comprises1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC) and/or whereinthe hydrophilic polymer comprises ethylene glycol or polyethylene glycol(PEG); and/or wherein the particle further comprises cholesterol (e.g.,particle from formulation 1=DOTAP 100, DMPC 0, PEG 0, Cholesterol 0;formulation number 2=DOTAP 90, DMPC 0, PEG 10, Cholesterol 0;formulation number 3=DOTAP 90, DMPC 0, PEG 5, Cholesterol 5), whereinparticles are formed using an efficient, multistep process whereinfirst, effector protein and RNA are mixed together, e.g., at a 1:1 molarratio, e.g., at room temperature (e.g., for 30 minutes, e.g., insterile, nuclease free 1×PBS; and separately, DOTAP, DMPC, PEG, andcholesterol as applicable for the formulation are dissolved in alcohol,e.g., 100% ethanol; and, the two solutions are mixed together to formparticles containing the complexes).

Nucleic acid-targeting effector proteins (such as a Type VI protein suchas C2c2) mRNA and guide RNA may be delivered simultaneously usingparticles or lipid envelopes.

For example, Su X, Fricke J, Kavanagh D G, Irvine D J (“In vitro and invivo mRNA delivery using lipid-enveloped pH-responsive polymernanoparticles” Mol Pharm. 2011 Jun. 6; 8(3):774-87. doi:10.1021/mp100390w. Epub 2011 Apr. 1) describes biodegradable core-shellstructured particles with a poly(O-amino ester) (PBAE) core enveloped bya phospholipid bilayer shell. These were developed for in vivo mRNAdelivery. The pH-responsive PBAE component was chosen to promoteendosome disruption, while the lipid surface layer was selected tominimize toxicity of the polycation core. Such are, therefore, preferredfor delivering RNA of the present invention.

In one embodiment, particles based on self-assembling bioadhesivepolymers are contemplated, which may be applied to oral delivery ofpeptides, intravenous delivery of peptides and nasal delivery ofpeptides, all to the brain. Other embodiments, such as oral absorptionand ocular delivery of hydrophobic drugs are also contemplated. Themolecular envelope technology involves an engineered polymer envelopewhich is protected and delivered to the site of the disease (see, e.g.,Mazza, M. et al. ACS Nano, 2013. 7(2): 1016-1026; Siew, A., et al. MolPharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012.161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80;Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N. L., etal. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N. L., et al. J RamanSpect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface2010. 7:S423-33; Uchegbu, I. F. Expert Opin Drug Deliv, 2006.3(5):629-40; Qu, X., et al. Biomacromolecules, 2006. 7(12):3452-9 andUchegbu, I. F., et al. Int J Pharm, 2001.224:185-199). Doses of about 5mg/kg are contemplated, with single or multiple doses, depending on thetarget tissue.

In one embodiment, particles that can deliver RNA to a cancer cell tostop tumor growth developed by Dan Anderson's lab at MIT may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention. In particular, the Anderson lab developed fully automated,combinatorial systems for the synthesis, purification, characterization,and formulation of new biomaterials and nanoformulations. See, e.g.,Alabi et al., Proc Natl Acad Sci USA. 2013 Aug. 6; 110(32):12881-6;Zhang et al., Adv Mater. 2013 Sep. 6; 25(33):4641-5; Jiang et al., NanoLett. 2013 Mar. 13; 13(3):1059-64; Karagiannis et al., ACS Nano. 2012Oct. 23; 6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug. 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun. 3; 7(6):389-93.

US patent application 20110293703 relates to lipidoid compounds are alsoparticularly useful in the administration of polynucleotides, which maybe applied to deliver the nucleic acid-targeting system of the presentinvention. In one aspect, the aminoalcohol lipidoid compounds arecombined with an agent to be delivered to a cell or a subject to formmicroparticles, nanoparticles, liposomes, or micelles. The agent to bedelivered by the particles, liposomes, or micelles may be in the form ofa gas, liquid, or solid, and the agent may be a polynucleotide, protein,peptide, or small molecule. The aminoalcohol lipidoid compounds may becombined with other aminoalcohol lipidoid compounds, polymers (syntheticor natural), surfactants, cholesterol, carbohydrates, proteins, lipids,etc. to form the particles. These particles may then optionally becombined with a pharmaceutical excipient to form a pharmaceuticalcomposition.

US Patent Publication No. 20110293703 also provides methods of preparingthe aminoalcohol lipidoid compounds. One or more equivalents of an amineare allowed to react with one or more equivalents of anepoxide-terminated compound under suitable conditions to form anaminoalcohol lipidoid compound of the present invention. In certainembodiments, all the amino groups of the amine are fully reacted withthe epoxide-terminated compound to form tertiary amines. In otherembodiments, all the amino groups of the amine are not fully reactedwith the epoxide-terminated compound to form tertiary amines therebyresulting in primary or secondary amines in the aminoalcohol lipidoidcompound. These primary or secondary amines are left as is or may bereacted with another electrophile such as a different epoxide-terminatedcompound. As will be appreciated by one skilled in the art, reacting anamine with less than excess of epoxide-terminated compound will resultin a plurality of different aminoalcohol lipidoid compounds with variousnumbers of tails. Certain amines may be fully functionalized with twoepoxide-derived compound tails while other molecules will not becompletely functionalized with epoxide-derived compound tails. Forexample, a diamine or polyamine may include one, two, three, or fourepoxide-derived compound tails off the various amino moieties of themolecule resulting in primary, secondary, and tertiary amines. Incertain embodiments, all the amino groups are not fully functionalized.In certain embodiments, two of the same types of epoxide-terminatedcompounds are used. In other embodiments, two or more differentepoxide-terminated compounds are used. The synthesis of the aminoalcohollipidoid compounds is performed with or without solvent, and thesynthesis may be performed at higher temperatures ranging from 30-100°C., preferably at approximately 50-90° C. The prepared aminoalcohollipidoid compounds may be optionally purified. For example, the mixtureof aminoalcohol lipidoid compounds may be purified to yield anaminoalcohol lipidoid compound with a particular number ofepoxide-derived compound tails. Or the mixture may be purified to yielda particular stereo- or regioisomer. The aminoalcohol lipidoid compoundsmay also be alkylated using an alkyl halide (e.g., methyl iodide) orother alkylating agent, and/or they may be acylated.

US Patent Publication No. 20110293703 also provides libraries ofaminoalcohol lipidoid compounds prepared by the inventive methods. Theseaminoalcohol lipidoid compounds may be prepared and/or screened usinghigh-throughput techniques involving liquid handlers, robots, microtiterplates, computers, etc. In certain embodiments, the aminoalcohollipidoid compounds are screened for their ability to transfectpolynucleotides or other agents (e.g., proteins, peptides, smallmolecules) into the cell.

US Patent Publication No. 20130302401 relates to a class ofpoly(beta-amino alcohols) (PBAAs) has been prepared using combinatorialpolymerization. The inventive PBAAs may be used in biotechnology andbiomedical applications as coatings (such as coatings of films ormultilayer films for medical devices or implants), additives, materials,excipients, non-biofouling agents, micropatterning agents, and cellularencapsulation agents. When used as surface coatings, these PBAAselicited different levels of inflammation, both in vitro and in vivo,depending on their chemical structures. The large chemical diversity ofthis class of materials allowed us to identify polymer coatings thatinhibit macrophage activation in vitro. Furthermore, these coatingsreduce the recruitment of inflammatory cells, and reduce fibrosis,following the subcutaneous implantation of carboxylated polystyrenemicroparticles. These polymers may be used to form polyelectrolytecomplex capsules for cell encapsulation. The invention may also havemany other biological applications such as antimicrobial coatings, DNAor siRNA delivery, and stem cell tissue engineering. The teachings of USPatent Publication No. 20130302401 may be applied to the nucleicacid-targeting system of the present invention.

In another embodiment, lipid nanoparticles (LNPs) are contemplated. Ananti-transthyretin small interfering RNA has been encapsulated in lipidnanoparticles and delivered to humans (see, e.g., Coelho et al., N EnglJ Med 2013; 369:819-29), and such a system may be adapted and applied tothe nucleic acid-targeting system of the present invention. Doses ofabout 0.01 to about 1 mg per kg of body weight administeredintravenously are contemplated. Medications to reduce the risk ofinfusion-related reactions are contemplated, such as dexamethasone,acetaminophen, diphenhydramine or cetirizine, and ranitidine arecontemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeksfor five doses are also contemplated.

LNPs have been shown to be highly effective in delivering siRNAs to theliver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol.3, No. 4, pages 363-470) and are therefore contemplated for deliveringRNA encoding nucleic acid-targeting effector protein to the liver. Adosage of about four doses of 6 mg/kg of the LNP every two weeks may becontemplated. Tabernero et al. demonstrated that tumor regression wasobserved after the first 2 cycles of LNPs dosed at 0.7 mg/kg, and by theend of 6 cycles the patient had achieved a partial response withcomplete regression of the lymph node metastasis and substantialshrinkage of the liver tumors. A complete response was obtained after 40doses in this patient, who has remained in remission and completedtreatment after receiving doses over 26 months. Two patients with RCCand extrahepatic sites of disease including kidney, lung, and lymphnodes that were progressing following prior therapy with VEGF pathwayinhibitors had stable disease at all sites for approximately 8 to 12months, and a patient with PNET and liver metastases continued on theextension study for 18 months (36 doses) with stable disease.

However, the charge of the LNP must be taken into consideration. Ascationic lipids combined with negatively charged lipids to inducenonbilayer structures that facilitate intracellular delivery. Becausecharged LNPs are rapidly cleared from circulation following intravenousinjection, ionizable cationic lipids with pKa values below 7 weredeveloped (see, e.g., Rosin et al., Molecular Therapy, vol. 19, no. 12,pages 1286-2200, December 2011). Negatively charged polymers such as RNAmay be loaded into LNPs at low pH values (e.g., pH 4) where theionizable lipids display a positive charge. However, at physiological pHvalues, the LNPs exhibit a low surface charge compatible with longercirculation times. Four species of ionizable cationic lipids have beenfocused upon, namely 1,2-Bis(linoleoyloxy)-3-(dimethylamino)propane(DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).It has been shown that LNP siRNA systems containing these lipids exhibitremarkably different gene silencing properties in hepatocytes in vivo,with potencies varying according to the seriesDLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII genesilencing model (see, e.g., Rosin et al., Molecular Therapy, vol. 19,no. 12, pages 1286-2200, December 2011). A dosage of 1 μg/ml of LNP orCRISPR-Cas RNA in or associated with the LNP may be contemplated,especially for a formulation containing DLinKC2-DMA.

Preparation of LNPs and CRISPR-Cas encapsulation may be used and/oradapted from Rosin et al., Molecular Therapy, vol. 19, no. 12, pages1286-2200, December 2011). The cationic lipids1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP),1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA),1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA),1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA),(3-o-[2″-(methoxypolyethyleneglycol)2000)succinoyl]-1,2-dimyristoyl-sn-glycol(PEG-S-DMG), and R-3-[(ω-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropyl-3-amine (PEG-C-DOMG) may be providedby Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized.Cholesterol may be purchased from Sigma (St Louis, Mo.). The specificnucleic acid-targeting complex (CRISPR-Cas) RNA may be encapsulated inLNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationiclipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may beincorporated to assess cellular uptake, intracellular delivery, andbiodistribution. Encapsulation may be performed by dissolving lipidmixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG(40:10:40:10 molar ratio) in ethanol to a final lipid concentration of10 mmol/1. This ethanol solution of lipid may be added drop-wise to 50mmol/1 citrate, pH 4.0 to form multilamellar vesicles to produce a finalconcentration of 30% ethanol vol/vol. Large unilamellar vesicles may beformed following extrusion of multilamellar vesicles through two stacked80 nm Nuclepore polycarbonate filters using the Extruder (NorthernLipids, Vancouver, Canada). Encapsulation may be achieved by adding RNAdissolved at 2 mg/ml in 50 mmol/1 citrate, pH 4.0 containing 30% ethanolvol/vol drop-wise to extruded preformed large unilamellar vesicles andincubation at 31° C. for 30 minutes with constant mixing to a finalRNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol andneutralization of formulation buffer were performed by dialysis againstphosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2regenerated cellulose dialysis membranes. Particle size distribution maybe determined by dynamic light scattering using a NICOMP 370 particlesizer, the vesicle/intensity modes, and Gaussian fitting (NicompParticle Sizing, Santa Barbara, Calif.). The particle size for all threeLNP systems may be ˜70 nm in diameter. RNA encapsulation efficiency maybe determined by removal of free RNA using Viva®PureD MiniH columns(Sartorius Stedim Biotech) from samples collected before and afterdialysis. The encapsulated RNA may be extracted from the elutedparticles and quantified at 260 nm. RNA to lipid ratio was determined bymeasurement of cholesterol content in vesicles using the Cholesterol Eenzymatic assay from Wako Chemicals USA (Richmond, Va.). In conjunctionwith the herein discussion of LNPs and PEG lipids, PEGylated liposomesor LNPs are likewise suitable for delivery of a nucleic acid-targetingsystem or components thereof.

Preparation of large LNPs may be used and/or or adapted from Rosin etal., Molecular Therapy, vol. 19, no. 12, pages 1286-2200, December 2011.A lipid premix solution (20.4 mg/ml total lipid concentration) may beprepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premixat a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids maybe subsequently hydrated by combining the mixture with 1.85 volumes ofcitrate buffer (10 mmol/1, pH 3.0) with vigorous stirring, resulting inspontaneous liposome formation in aqueous buffer containing 35% ethanol.The liposome solution may be incubated at 37° C. to allow fortime-dependent increase in particle size. Aliquots may be removed atvarious times during incubation to investigate changes in liposome sizeby dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments,Worcestershire, UK). Once the desired particle size is achieved, anaqueous PEG lipid solution (stock=10 mg/ml PEG-DMG in 35% (vol/vol)ethanol) may be added to the liposome mixture to yield a final PEG molarconcentration of 3.5% of total lipid. Upon addition of PEG-lipids, theliposomes should their size, effectively quenching further growth. RNAmay then be added to the empty liposomes at a RNA to total lipid ratioof approximately 1:10 (wt:wt), followed by incubation for 30 minutes at37° C. to form loaded LNPs. The mixture may be subsequently dialyzedovernight in PBS and filtered with a 0.45 m syringe filter.

Spherical Nucleic Acid (SNA™) constructs and other particles(particularly gold particles) are also contemplated as a means todeliver nucleic acid-targeting system to intended targets. Significantdata show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™)constructs, based upon nucleic acid-functionalized gold particles, areuseful.

Literature that may be employed in conjunction with herein teachingsinclude: Cutler et al., J. Am. Chem. Soc. 2011 133:9254-9257, Hao etal., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970,Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., NanoLett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012109:11975-80, Mirkin, Nanomedicine 2012 7:635-638, Zhang et al., J. Am.Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choiet al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen etal., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small,10:186-192.

Self-assembling particles with RNA may be constructed withpolyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD)peptide ligand attached at the distal end of the polyethylene glycol(PEG). This system has been used, for example, as a means to targettumor neovasculature expressing integrins and deliver siRNA inhibitingvascular endothelial growth factor receptor-2 (VEGFR2) expression andthereby achieve tumor angiogenesis (see, e.g., Schiffelers et al.,Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may beprepared by mixing equal volumes of aqueous solutions of cationicpolymer and nucleic acid to give a net molar excess of ionizablenitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.The electrostatic interactions between cationic polymers and nucleicacid resulted in the formation of polyplexes with average particle sizedistribution of about 100 nm, hence referred to here as nanoplexes. Adosage of about 100 to 200 mg of nucleic acid-targeting complex RNA isenvisioned for delivery in the self-assembling particles of Schiffelerset al.

The nanoplexes of Bartlett et al. (PNAS, Sep. 25, 2007, vol. 104, no.39) may also be applied to the present invention. The nanoplexes ofBartlett et al. are prepared by mixing equal volumes of aqueoussolutions of cationic polymer and nucleic acid to give a net molarexcess of ionizable nitrogen (polymer) to phosphate (nucleic acid) overthe range of 2 to 6. The electrostatic interactions between cationicpolymers and nucleic acid resulted in the formation of polyplexes withaverage particle size distribution of about 100 nm, hence referred tohere as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized asfollows: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acidmono(N-hydroxysuccinimide ester) (DOTA-NHS ester) was ordered fromMacrocyclics (Dallas, Tex.). The amine modified RNA sense strand with a100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) wasadded to a microcentrifuge tube. The contents were reacted by stirringfor 4 h at room temperature. The DOTA-RNAsense conjugate wasethanol-precipitated, resuspended in water, and annealed to theunmodified antisense strand to yield DOTA-siRNA. All liquids werepretreated with Chelex-100 (Bio-Rad, Hercules, Calif.) to remove tracemetal contaminants. Tf-targeted and nontargeted siRNA particles may beformed by using cyclodextrin-containing polycations. Typically,particles were formed in water at a charge ratio of 3 (+/−) and an siRNAconcentration of 0.5 g/liter. One percent of the adamantane-PEGmolecules on the surface of the targeted particles were modified with Tf(adamantane-PEG-Tf). The particles were suspended in a 5% (wt/vol)glucose carrier solution for injection.

Davis et al. (Nature, Vol 464, 15 Apr. 2010) conducts a RNA clinicaltrial that uses a targeted particle-delivery system (clinical trialregistration number NCT00689065). Patients with solid cancers refractoryto standard-of-care therapies are administered doses of targetedparticles on days 1, 3, 8 and 10 of a 21-day cycle by a 30-minintravenous infusion. The particles comprise, consist essentially of, orconsist of a synthetic delivery system containing: (1) a linear,cyclodextrin-based polymer (CDP), (2) a human transferrin protein (TF)targeting ligand displayed on the exterior of the nanoparticle to engageTF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilicpolymer (polyethylene glycol (PEG) used to promote nanoparticlestability in biological fluids), and (4) siRNA designed to reduce theexpression of the RRM2 (sequence used in the clinic was previouslydenoted siR2B+5). The TFR has long been known to be upregulated inmalignant cells, and RRM2 is an established anti-cancer target. Theseparticles (clinical version denoted as CALAA-01) have been shown to bewell tolerated in multi-dosing studies in non-human primates. Although asingle patient with chronic myeloid leukaemia has been administeredsiRNA by liposomal delivery, Davis et al.'s clinical trial is theinitial human trial to systemically deliver siRNA with a targeteddelivery system and to treat patients with solid cancer. To ascertainwhether the targeted delivery system can provide effective delivery offunctional siRNA to human tumours, Davis et al. investigated biopsiesfrom three patients from three different dosing cohorts; patients A, Band C, all of whom had metastatic melanoma and received CALAA-01 dosesof 18, 24 and 30 mg m⁻² siRNA, respectively. Similar doses may also becontemplated for the nucleic acid-targeting system of the presentinvention. The delivery of the invention may be achieved with particlescontaining a linear, cyclodextrin-based polymer (CDP), a humantransferrin protein (TF) targeting ligand displayed on the exterior ofthe particle to engage TF receptors (TFR) on the surface of the cancercells and/or a hydrophilic polymer (for example, polyethylene glycol(PEG) used to promote particle stability in biological fluids).

In terms of this invention, it is preferred to have one or morecomponents of nucleic acid-targeting complex, e.g., nucleicacid-targeting effector protein or mRNA, or guide RNA delivered usingparticles or lipid envelopes. Other delivery systems or vectors are maybe used in conjunction with the particle aspects of the invention.

In general, a “nanoparticle” refers to any particle having a diameter ofless than 1000 nm. In certain preferred embodiments, nanoparticles ofthe invention have a greatest dimension (e.g., diameter) of 500 nm orless. In other preferred embodiments, nanoparticles of the inventionhave a greatest dimension ranging between 25 nm and 200 nm. In otherpreferred embodiments, particles of the invention have a greatestdimension of 100 nm or less. In other preferred embodiments,nanoparticles of the invention have a greatest dimension ranging between35 nm and 60 nm.

Particles encompassed in the present invention may be provided indifferent forms, e.g., as solid particles (e.g., metal such as silver,gold, iron, titanium), non-metal, lipid-based solids, polymers),suspensions of particles, or combinations thereof. Metal, dielectric,and semiconductor particles may be prepared, as well as hybridstructures (e.g., core-shell particles). Particles made ofsemiconducting material may also be labeled quantum dots if they aresmall enough (typically sub 10 nm) that quantization of electronicenergy levels occurs. Such nanoscale particles are used in biomedicalapplications as drug carriers or imaging agents and may be adapted forsimilar purposes in the present invention.

Semi-solid and soft particles have been manufactured, and are within thescope of the present invention. A prototype particle of semi-solidnature is the liposome. Various types of liposome particles arecurrently used clinically as delivery systems for anticancer drugs andvaccines. Particles with one half hydrophilic and the other halfhydrophobic are termed Janus particles and are particularly effectivefor stabilizing emulsions. They can self-assemble at water/oilinterfaces and act as solid surfactants.

U.S. Pat. No. 8,709,843, incorporated herein by reference, provides adrug delivery system for targeted delivery of therapeuticagent-containing particles to tissues, cells, and intracellularcompartments. The invention provides targeted particles comprisingpolymer conjugated to a surfactant, hydrophilic polymer or lipid.

U.S. Pat. No. 6,007,845, incorporated herein by reference, providesparticles which have a core of a multiblock copolymer formed bycovalently linking a multifunctional compound with one or morehydrophobic polymers and one or more hydrophilic polymers, and contain abiologically active material.

U.S. Pat. No. 5,855,913, incorporated herein by reference, provides aparticulate composition having aerodynamically light particles having atap density of less than 0.4 g/cm3 with a mean diameter of between 5 μmand 30 μm, incorporating a surfactant on the surface thereof for drugdelivery to the pulmonary system.

U.S. Pat. No. 5,985,309, incorporated herein by reference, providesparticles incorporating a surfactant and/or a hydrophilic or hydrophobiccomplex of a positively or negatively charged therapeutic or diagnosticagent and a charged molecule of opposite charge for delivery to thepulmonary system.

U.S. Pat. No. 5,543,158, incorporated herein by reference, providesbiodegradable injectable particles having a biodegradable solid corecontaining a biologically active material and poly(alkylene glycol)moieties on the surface.

WO2012135025 (also published as US20120251560), incorporated herein byreference, describes conjugated polyethyleneimine (PEI) polymers andconjugated aza-macrocycles (collectively referred to as “conjugatedlipomer” or “lipomers”). In certain embodiments, it can be envisionedthat such methods and materials of herein-cited documents, e.g.,conjugated lipomers can be used in the context of the nucleicacid-targeting system to achieve in vitro, ex vivo and in vivo genomicperturbations to modify gene expression, including modulation of proteinexpression.

In one embodiment, the particle may be epoxide-modified lipid-polymer,advantageously 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al.Nature Nanotechnology (2014) published online 11 May 2014,doi:10.1038/nnano.2014.84). C71 was synthesized by reacting C15epoxide-terminated lipids with PEI600 at a 14:1 molar ratio, and wasformulated with C14 PEG2000 to produce particles (diameter between 35and 60 nm) that were stable in PBS solution for at least 40 days.

An epoxide-modified lipid-polymer may be utilized to deliver the nucleicacid-targeting system of the present invention to pulmonary,cardiovascular or renal cells, however, one of skill in the art mayadapt the system to deliver to other target organs. Dosage ranging fromabout 0.05 to about 0.6 mg/kg are envisioned. Dosages over several daysor weeks are also envisioned, with a total dosage of about 2 mg/kg.

Exosomes

Exosomes are endogenous nano-vesicles that transport RNAs and proteins,and which can deliver RNA to the brain and other target organs. Toreduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used self-derived dendritic cells for exosome production. Targetingto the brain was achieved by engineering the dendritic cells to expressLamp2b, an exosomal membrane protein, fused to the neuron-specific RVGpeptide. Purified exosomes were loaded with exogenous RNA byelectroporation. Intravenously injected RVG-targeted exosomes deliveredGAPDH siRNA specifically to neurons, microglia, oligodendrocytes in thebrain, resulting in a specific gene knockdown. Pre-exposure to RVGexosomes did not attenuate knockdown, and non-specific uptake in othertissues was not observed. The therapeutic potential of exosome-mediatedsiRNA delivery was demonstrated by the strong mRNA (60%) and protein(62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To obtain a pool of immunologically inert exosomes, Alvarez-Erviti etal. harvested bone marrow from inbred C57BL/6 mice with a homogenousmajor histocompatibility complex (MHC) haplotype. As immature dendriticcells produce large quantities of exosomes devoid of T-cell activatorssuch as MHC-II and CD86, Alvarez-Erviti et al. selected for dendriticcells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for7 d. Exosomes were purified from the culture supernatant the followingday using well-established ultracentrifugation protocols. The exosomesproduced were physically homogenous, with a size distribution peaking at80 nm in diameter as determined by particle tracking analysis (NTA) andelectron microscopy. Alvarez-Erviti et al. obtained 6-12 μg of exosomes(measured based on protein concentration) per 10⁶ cells.

Next, Alvarez-Erviti et al. investigated the possibility of loadingmodified exosomes with exogenous cargoes using electroporation protocolsadapted for nanoscale applications. As electroporation for membraneparticles at the nanometer scale is not well-characterized, nonspecificCy5-labeled RNA was used for the empirical optimization of theelectroporation protocol. The amount of encapsulated RNA was assayedafter ultracentrifugation and lysis of exosomes. Electroporation at 400V and 125 μF resulted in the greatest retention of RNA and was used forall subsequent experiments.

Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNAencapsulated in 150 μg of RVG exosomes to normal C57BL/6 mice andcompared the knockdown efficiency to four controls: untreated mice, miceinjected with RVG exosomes only, mice injected with BACE1 siRNAcomplexed to an in vivo cationic liposome reagent and mice injected withBACE1 siRNA complexed to RVG-9R, the RVG peptide conjugated to 9D-arginines that electrostatically binds to the siRNA. Cortical tissuesamples were analyzed 3D after administration and a significant proteinknockdown (45%, P<0.05, versus 62%, P<0.01) in both siRNA-RVG-9R-treatedand siRNA-RVG exosome-treated mice was observed, resulting from asignificant decrease in BACE1 mRNA levels (66% [+ or −] 15%, P<0.001 and61% [+ or −] 13% respectively, P<0.01). Moreover, Applicantsdemonstrated a significant decrease (55%, P<0.05) in the total[beta]-amyloid 1-42 levels, a main component of the amyloid plaques inAlzheimer's pathology, in the RVG-exosome-treated animals. The decreaseobserved was greater than the β-amyloid 1-40 decrease demonstrated innormal mice after intraventricular injection of BACE1 inhibitors.Alvarez-Erviti et al. carried out 5′-rapid amplification of cDNA ends(RACE) on BACE1 cleavage product, which provided evidence ofRNAi-mediated knockdown by the siRNA.

Finally, Alvarez-Erviti et al. investigated whether RNA-RVG exosomesinduced immune responses in vivo by assessing IL-6, IP-10, TNFα andIFN-α serum concentrations. Following exosome treatment, nonsignificantchanges in all cytokines were registered similar to siRNA-transfectionreagent treatment in contrast to siRNA-RVG-9R, which potently stimulatedIL-6 secretion, confirming the immunologically inert profile of theexosome treatment. Given that exosomes encapsulate only 20% of siRNA,delivery with RVG-exosome appears to be more efficient than RVG-9Rdelivery as comparable mRNA knockdown and greater protein knockdown wasachieved with fivefold less siRNA without the corresponding level ofimmune stimulation. This experiment demonstrated the therapeuticpotential of RVG-exosome technology, which is potentially suited forlong-term silencing of genes related to neurodegenerative diseases. Theexosome delivery system of Alvarez-Erviti et al. may be applied todeliver the nucleic acid-targeting system of the present invention totherapeutic targets, especially neurodegenerative diseases. A dosage ofabout 100 to 1000 mg of nucleic acid-targeting system encapsulated inabout 100 to 1000 mg of RVG exosomes may be contemplated for the presentinvention.

El-Andaloussi et al. (Nature Protocols 7, 2112-2126(2012)) discloses howexosomes derived from cultured cells can be harnessed for delivery ofRNA in vitro and in vivo. This protocol first describes the generationof targeted exosomes through transfection of an expression vector,comprising an exosomal protein fused with a peptide ligand. Next,El-Andaloussi et al. explain how to purify and characterize exosomesfrom transfected cell supernatant. Next, El-Andaloussi et al. detailcrucial steps for loading RNA into exosomes. Finally, El-Andaloussi etal. outline how to use exosomes to efficiently deliver RNA in vitro andin vivo in mouse brain. Examples of anticipated results in whichexosome-mediated RNA delivery is evaluated by functional assays andimaging are also provided. The entire protocol takes ˜3 weeks. Deliveryor administration according to the invention may be performed usingexosomes produced from self-derived dendritic cells. From the hereinteachings, this can be employed in the practice of the invention.

In another embodiment, the plasma exosomes of Wahlgren et al. (NucleicAcids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomesare nano-sized vesicles (30-90 nm in size) produced by many cell types,including dendritic cells (DC), B cells, T cells, mast cells, epithelialcells and tumor cells. These vesicles are formed by inward budding oflate endosomes and are then released to the extracellular environmentupon fusion with the plasma membrane. Because exosomes naturally carryRNA between cells, this property may be useful in gene therapy, and fromthis disclosure can be employed in the practice of the instantinvention.

Exosomes from plasma can be prepared by centrifugation of buffy coat at900 g for 20 min to isolate the plasma followed by harvesting cellsupernatants, centrifuging at 300 g for 10 min to eliminate cells and at16 500 g for 30 min followed by filtration through a 0.22 mm filter.Exosomes are pelleted by ultracentrifugation at 120 000 g for 70 min.Chemical transfection of siRNA into exosomes is carried out according tothe manufacturer's instructions in RNAi Human/Mouse Starter Kit (Qiagen,Hilden, Germany). siRNA is added to 100 ml PBS at a final concentrationof 2 mmol/ml. After adding HiPerFect transfection reagent, the mixtureis incubated for 10 min at RT. In order to remove the excess ofmicelles, the exosomes are re-isolated using aldehyde/sulfate latexbeads. The chemical transfection of nucleic acid-targeting system intoexosomes may be conducted similarly to siRNA. The exosomes may beco-cultured with monocytes and lymphocytes isolated from the peripheralblood of healthy donors. Therefore, it may be contemplated that exosomescontaining nucleic acid-targeting system may be introduced to monocytesand lymphocytes of and autologously reintroduced into a human.Accordingly, delivery or administration according to the invention maybe performed using plasma exosomes.

Liposomes

Delivery or administration according to the invention can be performedwith liposomes. Liposomes are spherical vesicle structures composed of auni- or multilamellar lipid bilayer surrounding internal aqueouscompartments and a relatively impermeable outer lipophilic phospholipidbilayer. Liposomes have gained considerable attention as drug deliverycarriers because they are biocompatible, nontoxic, can deliver bothhydrophilic and lipophilic drug molecules, protect their cargo fromdegradation by plasma enzymes, and transport their load acrossbiological membranes and the blood brain barrier (BBB) (see, e.g., Spuchand Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12pages, 2011. doi:10.1155/2011/469679 for review).

Liposomes can be made from several different types of lipids; however,phospholipids are most commonly used to generate liposomes as drugcarriers. Although liposome formation is spontaneous when a lipid filmis mixed with an aqueous solution, it can also be expedited by applyingforce in the form of shaking by using a homogenizer, sonicator, or anextrusion apparatus (see, e.g., Spuch and Navarro, Journal of DrugDelivery, vol. 2011, Article ID 469679, 12 pages, 2011.doi:10.1155/2011/469679 for review).

Several other additives may be added to liposomes in order to modifytheir structure and properties. For instance, either cholesterol orsphingomyelin may be added to the liposomal mixture in order to helpstabilize the liposomal structure and to prevent the leakage of theliposomal inner cargo. Further, liposomes are prepared from hydrogenatedegg phosphatidylcholine or egg phosphatidylcholine, cholesterol, anddicetyl phosphate, and their mean vesicle sizes were adjusted to about50 and 100 nm (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

A liposome formulation may be mainly comprised of natural phospholipidsand lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidyl choline(DSPC), sphingomyelin, egg phosphatidylcholines andmonosialoganglioside. Since this formulation is made up of phospholipidsonly, liposomal formulations have encountered many challenges, one ofthe ones being the instability in plasma. Several attempts to overcomethese challenges have been made, specifically in the manipulation of thelipid membrane. One of these attempts focused on the manipulation ofcholesterol. Addition of cholesterol to conventional formulationsreduces rapid release of the encapsulated bioactive compound into theplasma or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increasesthe stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery,vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679for review).

In a particularly advantageous embodiment, Trojan Horse liposomes (alsoknown as Molecular Trojan Horses) are desirable and protocols may befound at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long.These particles allow delivery of a transgene to the entire brain afteran intravascular injection. Without being bound by limitation, it isbelieved that neutral lipid particles with specific antibodiesconjugated to surface allow crossing of the blood brain barrier viaendocytosis. Applicant postulates utilizing Trojan Horse Liposomes todeliver the CRISPR family of nucleases to the brain via an intravascularinjection, which would allow whole brain transgenic animals without theneed for embryonic manipulation. About 1-5 g of DNA or RNA may becontemplated for in vivo administration in liposomes.

In another embodiment, the nucleic acid-targeting system or componentsthereof may be administered in liposomes, such as a stablenucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., NatureBiotechnology, Vol. 23, No. 8, August 2005). Daily intravenousinjections of about 1, 3 or 5 mg/kg/day of a specific nucleicacid-targeting system targeted in a SNALP are contemplated. The dailytreatment may be over about three days and then weekly for about fiveweeks. In another embodiment, a specific nucleic acid-targeting systemencapsulated SNALP administered by intravenous injection to at doses ofabout 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al.,Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may containthe lipids 3-N-[(o-methoxypoly(ethylene glycol) 2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar percent ratio (see, e.g., Zimmerman et al., NatureLetters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic-acid-lipid particles (SNALPs) haveproven to be effective delivery molecules to highly vascularizedHepG2-derived liver tumors but not in poorly vascularized HCT-116derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780).The SNALP liposomes may be prepared by formulating D-Lin-DMA andPEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol andsiRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio ofCholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes areabout 80-100 nm in size.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich, St Louis, Mo., USA), dipalmitoylphosphatidylcholine(Avanti Polar Lipids, Alabaster, Ala., USA), 3-N-[(o-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine, andcationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g.,Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kgtotal nucleic acid-targeting system per dose administered as, forexample, a bolus intravenous infusion may be contemplated.

In yet another embodiment, a SNALP may comprise synthetic cholesterol(Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC;Avanti Polar Lipids Inc.), PEG-cDMA, and1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA) (see, e.g.,Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for invivo studies may comprise a final lipid/RNA mass ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros andGollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug DeliveryReviews 64 (2012) 1730-1737). The stable nucleic acid lipid particle(SNALP) is comprised of four different lipids an ionizable lipid(DLinDMA) that is cationic at low pH, a neutral helper lipid,cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. Theparticle is approximately 80 nm in diameter and is charge-neutral atphysiologic pH. During formulation, the ionizable lipid serves tocondense lipid with the anionic RNA during particle formation. Whenpositively charged under increasingly acidic endosomal conditions, theionizable lipid also mediates the fusion of SNALP with the endosomalmembrane enabling release of RNA into the cytoplasm. The PEG-lipidstabilizes the particle and reduces aggregation during formulation, andsubsequently provides a neutral hydrophilic exterior that improvespharmacokinetic properties.

To date, two clinical programs have been initiated using SNALPformulations with RNA. Tekmira Pharmaceuticals recently completed aphase I single-dose study of SNALP-ApoB in adult volunteers withelevated LDL cholesterol. ApoB is predominantly expressed in the liverand jejunum and is essential for the assembly and secretion of VLDL andLDL. Seventeen subjects received a single dose of SNALP-ApoB (doseescalation across 7 dose levels). There was no evidence of livertoxicity (anticipated as the potential dose-limiting toxicity based onpreclinical studies). One (of two) subjects at the highest doseexperienced flu-like symptoms consistent with immune system stimulation,and the decision was made to conclude the trial.

Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employsthe SNALP technology described above and targets hepatocyte productionof both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). ThreeATTR syndromes have been described: familial amyloidotic polyneuropathy(FAP) and familial amyloidotic cardiomyopathy (FAC) both caused byautosomal dominant mutations in TTR; and senile systemic amyloidosis(SSA) cause by wildtype TTR. A placebo-controlled, singledose-escalation phase I trial of ALN-TTR01 was recently completed inpatients with ATTR. ALN-TTR01 was administered as a 15-minute IVinfusion to 31 patients (23 with study drug and 8 with placebo) within adose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatment was welltolerated with no significant increases in liver function tests.Infusion-related reactions were noted in 3 of 23 patients at ≥0.4 mg/kg;all responded to slowing of the infusion rate and all continued onstudy. Minimal and transient elevations of serum cytokines IL-6, IP-10and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (asanticipated from preclinical and NHP studies). Lowering of serum TTR,the expected pharmacodynamics effect of ALN-TTR01, was observed at 1mg/kg.

In yet another embodiment, a SNALP may be made by solubilizing acationic lipid, DSPC, cholesterol and PEG-lipid e.g., in ethanol, e.g.,at a molar ratio of 40:10:40:10, respectively (see, Semple et al.,Nature Biotechnology, Volume 28 Number 2 Feb. 2010, pp. 172-177). Thelipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) withmixing to a final ethanol and lipid concentration of 30% (vol/vol) and6.1 mg/ml, respectively, and allowed to equilibrate at 22° C. for 2 minbefore extrusion. The hydrated lipids were extruded through two stacked80 nm pore-sized filters (Nuclepore) at 22° C. using a Lipex Extruder(Northern Lipids) until a vesicle diameter of 70-90 nm, as determined bydynamic light scattering analysis, was obtained. This generally required1-3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueoussolution containing 30% ethanol) was added to the pre-equilibrated (35°C.) vesicles at a rate of ˜5 ml/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubatedfor a further 30 min at 35° C. to allow vesicle reorganization andencapsulation of the siRNA. The ethanol was then removed and theexternal buffer replaced with PBS (155 mM NaCl, 3 mM Na₂HIPO₄, 1 mMKH₂PO₄, pH 7.5) by either dialysis or tangential flow diafiltration.siRNA were encapsulated in SNALP using a controlled step-wise dilutionmethod process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA(cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti PolarLipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molarratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles,SNALP were dialyzed against PBS and filter sterilized through a 0.2 mfilter before use. Mean particle sizes were 75-85 nm and 90-95% of thesiRNA was encapsulated within the lipid particles. The final siRNA/lipidratio in formulations used for in vivo testing was ˜0.15 (wt/wt).LNP-siRNA systems containing Factor VII siRNA were diluted to theappropriate concentrations in sterile PBS immediately before use and theformulations were administered intravenously through the lateral tailvein in a total volume of 10 ml/kg. This method and these deliverysystems may be extrapolated to the nucleic acid-targeting system of thepresent invention.

Other Lipids

Other cationic lipids, such as amino lipid2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) maybe utilized to encapsulate nucleic acid-targeting system or componentsthereof or nucleic acid molecule(s) coding therefor e.g., similar toSiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533),and hence may be employed in the practice of the invention. A preformedvesicle with the following lipid composition may be contemplated: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and(R)-2,3-bis(octadecyloxy) propyl-1-(methoxy poly(ethyleneglycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10,respectively, and a FVII siRNA/total lipid ratio of approximately 0.05(w/w). To ensure a narrow particle size distribution in the range of70-90 nm and a low polydispersity index of 0.11±0.04 (n=56), theparticles may be extruded up to three times through 80 nm membranesprior to adding the guide RNA. Particles containing the highly potentamino lipid 16 may be used, in which the molar ratio of the four lipidcomponents 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) whichmay be further optimized to enhance in vivo activity.

Michael S D Kormann et al. (“Expression of therapeutic proteins afterdelivery of chemically modified mRNA in mice: Nature Biotechnology,Volume:29, Pages: 154-157 (2011)) describes the use of lipid envelopesto deliver RNA. Use of lipid envelopes is also preferred in the presentinvention.

In another embodiment, lipids may be formulated with the nucleicacid-targeting system of the present invention or component(s) thereofor nucleic acid molecule(s) coding therefor to form lipid nanoparticles(LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C₁₂₋₂₀₀and colipids distearoylphosphatidyl choline, cholesterol, and PEG-DMGmay be formulated with RNA-targeting system instead of siRNA (see, e.g.,Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4;doi:10.1038/mtna.2011.3) using a spontaneous vesicle formationprocedure. The component molar ratio may be about 50/10/38.5/1.5(DLin-KC2-DMA or C₁₂₋₂₀₀/distearoylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be˜12:1 and 9:1 in the case of DLin-KC2-DMA and C₁₂₋₂₀₀ lipid particles(LNPs), respectively. The formulations may have mean particle diametersof ˜80 nm with >90% entrapment efficiency. A 3 mg/kg dose may becontemplated.

Tekmira has a portfolio of approximately 95 patent families, in the U.S.and abroad, that are directed to various aspects of LNPs and LNPformulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069;8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263;7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos. 1766035;1519714; 1781593 and 1664316), all of which may be used and/or adaptedto the present invention.

The nucleic acid-targeting system or components thereof or nucleic acidmolecule(s) coding therefor may be delivered encapsulated in PLGAMicrospheres such as that further described in US published applications20130252281 and 20130245107 and 20130244279 (assigned to ModernaTherapeutics) which relate to aspects of formulation of compositionscomprising modified nucleic acid molecules which may encode a protein, aprotein precursor, or a partially or fully processed form of the proteinor a protein precursor. The formulation may have a molar ratio50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEGlipid). The PEG lipid may be selected from, but is not limited to,PEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrumet al., Delivery and Formulation of Engineered Nucleic Acids, USpublished application 20120251618.

Nanomerics' technology addresses bioavailability challenges for a broadrange of therapeutics, including low molecular weight hydrophobic drugs,peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).Specific administration routes for which the technology has demonstratedclear advantages include the oral route, transport across theblood-brain-barrier, delivery to solid tumours, as well as to the eye.See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb. 26; 7(2):1016-26;Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al.,2012, J Control Release. 2012 Jul. 20; 161(2):523-36.

US Patent Publication No. 20050019923 describes cationic dendrimers fordelivering bioactive molecules, such as polynucleotide molecules,peptides and polypeptides and/or pharmaceutical agents, to a mammalianbody. The dendrimers are suitable for targeting the delivery of thebioactive molecules to, for example, the liver, spleen, lung, kidney orheart (or even the brain). Dendrimers are synthetic 3-dimensionalmacromolecules that are prepared in a step-wise fashion from simplebranched monomer units, the nature and functionality of which can beeasily controlled and varied. Dendrimers are synthesized from therepeated addition of building blocks to a multifunctional core(divergent approach to synthesis), or towards a multifunctional core(convergent approach to synthesis) and each addition of a 3-dimensionalshell of building blocks leads to the formation of a higher generationof the dendrimers. Polypropyleneimine dendrimers start from adiaminobutane core to which is added twice the number of amino groups bya double Michael addition of acrylonitrile to the primary aminesfollowed by the hydrogenation of the nitriles. This results in adoubling of the amino groups. Polypropyleneimine dendrimers contain 100%protonable nitrogens and up to 64 terminal amino groups (generation 5,DAB 64). Protonable groups are usually amine groups which are able toaccept protons at neutral pH. The use of dendrimers as gene deliveryagents has largely focused on the use of the polyamidoamine andphosphorous containing compounds with a mixture of amine/amide orN—P(O₂)S as the conjugating units respectively with no work beingreported on the use of the lower generation polypropyleneiminedendrimers for gene delivery. Polypropyleneimine dendrimers have alsobeen studied as pH sensitive controlled release systems for drugdelivery and for their encapsulation of guest molecules when chemicallymodified by peripheral amino acid groups. The cytotoxicity andinteraction of polypropyleneimine dendrimers with DNA as well as thetransfection efficacy of DAB 64 has also been studied.

US Patent Publication No. 20050019923 is based upon the observationthat, contrary to earlier reports, cationic dendrimers, such aspolypropyleneimine dendrimers, display suitable properties, such asspecific targeting and low toxicity, for use in the targeted delivery ofbioactive molecules, such as genetic material. In addition, derivativesof the cationic dendrimer also display suitable properties for thetargeted delivery of bioactive molecules. See also, Bioactive Polymers,US published application 20080267903, which discloses “Various polymers,including cationic polyamine polymers and dendrimeric polymers, areshown to possess anti-proliferative activity, and may therefore beuseful for treatment of disorders characterised by undesirable cellularproliferation such as neoplasms and tumours, inflammatory disorders(including autoimmune disorders), psoriasis and atherosclerosis. Thepolymers may be used alone as active agents, or as delivery vehicles forother therapeutic agents, such as drug molecules or nucleic acids forgene therapy. In such cases, the polymers' own intrinsic anti-tumouractivity may complement the activity of the agent to be delivered.” Thedisclosures of these patent publications may be employed in conjunctionwith herein teachings for delivery of nucleic acid-targeting system(s)or component(s) thereof or nucleic acid molecule(s) coding therefor.

Supercharged Proteins

Supercharged proteins are a class of engineered or naturally occurringproteins with unusually high positive or negative net theoretical chargeand may be employed in delivery of nucleic acid-targeting system(s) orcomponent(s) thereof or nucleic acid molecule(s) coding therefor. Bothsuper negatively and super positively charged proteins exhibit aremarkable ability to withstand thermally or chemically inducedaggregation. Super positively charged proteins are also able topenetrate mammalian cells. Associating cargo with these proteins, suchas plasmid DNA, RNA, or other proteins, can enable the functionaldelivery of these macromolecules into mammalian cells both in vitro andin vivo. David Liu's lab reported the creation and characterization ofsupercharged proteins in 2007 (Lawrence et al., 2007, Journal of theAmerican Chemical Society 129, 10110-10112).

The nonviral delivery of RNA and plasmid DNA into mammalian cells arevaluable both for research and therapeutic applications (Akinc et al.,2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or othersuper positively charged protein) is mixed with RNAs in the appropriateserum-free media and allowed to complex prior addition to cells.Inclusion of serum at this stage inhibits formation of the superchargedprotein-RNA complexes and reduces the effectiveness of the treatment.The following protocol has been found to be effective for a variety ofcell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106,6111-6116). However, pilot experiments varying the dose of protein andRNA should be performed to optimize the procedure for specific celllines.

(1) One day before treatment, plate 1×10⁵ cells per well in a 48-wellplate.

(2) On the day of treatment, dilute purified +36 GFP protein inserum-free media to a final concentration 200 nM. Add RNA to a finalconcentration of 50 nM. Vortex to mix and incubate at room temperaturefor 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of +36 GFP and RNA, add the protein-RNAcomplexes to cells.

(5) Incubate cells with complexes at 37° C. for 4 h.

(6) Following incubation, aspirate the media and wash three times with20 U/mL heparin PBS. Incubate cells with serum-containing media for afurther 48 h or longer depending upon the assay for activity.

(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or otherappropriate method.

David Liu's lab has further found +36 GFP to be an effective plasmiddelivery reagent in a range of cells. As plasmid DNA is a larger cargothan siRNA, proportionately more +36 GFP protein is required toeffectively complex plasmids. For effective plasmid delivery Applicantshave developed a variant of +36 GFP bearing a C-terminal HA2 peptidetag, a known endosome-disrupting peptide derived from the influenzavirus hemagglutinin protein. The following protocol has been effectivein a variety of cells, but as above it is advised that plasmid DNA andsupercharged protein doses be optimized for specific cell lines anddelivery applications.

(1) One day before treatment, plate 1×10⁵ per well in a 48-well plate.

(2) On the day of treatment, dilute purified +36 GFP protein inserum-free media to a final concentration 2 mM. Add 1 mg of plasmid DNA.Vortex to mix and incubate at room temperature for 10 min.

(3) During incubation, aspirate media from cells and wash once with PBS.

(4) Following incubation of 36 GFP and plasmid DNA, gently add theprotein-DNA complexes to cells.

(5) Incubate cells with complexes at 370 C for 4 h.

(6) Following incubation, aspirate the media and wash with PBS. Incubatecells in serum-containing media and incubate for a further 24-48 h.

(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression)as appropriate.

See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106,6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752(2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011);Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods ofthe super charged proteins may be used and/or adapted for delivery ofthe nucleic acid-targeting system of the present invention. Thesesystems of Dr. Lui and documents herein in conjunction with hereinteachings can be employed in the delivery of nucleic acid-targetingsystem(s) or component(s) thereof or nucleic acid molecule(s) codingtherefor.

Cell Penetrating Peptides (CPPs)

In yet another embodiment, cell penetrating peptides (CPPs) arecontemplated for the delivery of the CRISPR Cas system. CPPs are shortpeptides that facilitate cellular uptake of various molecular cargo(from nanosize particles to small chemical molecules and large fragmentsof DNA). The term “cargo” as used herein includes but is not limited tothe group consisting of therapeutic agents, diagnostic probes, peptides,nucleic acids, antisense oligonucleotides, plasmids, proteins, particlesincluding nanoparticles, liposomes, chromophores, small molecules andradioactive materials. In aspects of the invention, the cargo may alsocomprise any component of the CRISPR Cas system or the entire functionalCRISPR Cas system. Aspects of the present invention further providemethods for delivering a desired cargo into a subject comprising: (a)preparing a complex comprising the cell penetrating peptide of thepresent invention and a desired cargo, and (b) orally, intraarticularly,intraperitoneally, intrathecally, intraarterially, intranasally,intraparenchymally, subcutaneously, intramuscularly, intravenously,dermally, intrarectally, or topically administering the complex to asubject. The cargo is associated with the peptides either throughchemical linkage via covalent bonds or through non-covalentinteractions.

The function of the CPPs are to deliver the cargo into cells, a processthat commonly occurs through endocytosis with the cargo delivered to theendosomes of living mammalian cells. Cell-penetrating peptides are ofdifferent sizes, amino acid sequences, and charges but all CPPs have onedistinct characteristic, which is the ability to translocate the plasmamembrane and facilitate the delivery of various molecular cargoes to thecytoplasm or an organelle. CPP translocation may be classified intothree main entry mechanisms: direct penetration in the membrane,endocytosis-mediated entry, and translocation through the formation of atransitory structure. CPPs have found numerous applications in medicineas drug delivery agents in the treatment of different diseases includingcancer and virus inhibitors, as well as contrast agents for celllabeling. Examples of the latter include acting as a carrier for GFP,MRI contrast agents, or quantum dots. CPPs hold great potential as invitro and in vivo delivery vectors for use in research and medicine.CPPs typically have an amino acid composition that either contains ahigh relative abundance of positively charged amino acids such as lysineor arginine or has sequences that contain an alternating pattern ofpolar/charged amino acids and non-polar, hydrophobic amino acids. Thesetwo types of structures are referred to as polycationic or amphipathic,respectively. A third class of CPPs are the hydrophobic peptides,containing only apolar residues, with low net charge or have hydrophobicamino acid groups that are crucial for cellular uptake. One of theinitial CPPs discovered was the trans-activating transcriptionalactivator (Tat) from Human Immunodeficiency Virus 1 (HIV-1) which wasfound to be efficiently taken up from the surrounding media by numerouscell types in culture. Since then, the number of known CPPs has expandedconsiderably and small molecule synthetic analogues with more effectiveprotein transduction properties have been generated. CPPs include butare not limited to Penetratin, Tat (48-60), Transportan, and (R-AhX-R)₄(Ahx=aminohexanoyl) (SEQ ID NO: 52).

U.S. Pat. No. 8,372,951, provides a CPP derived from eosinophil cationicprotein (ECP) which exhibits highly cell-penetrating efficiency and lowtoxicity. Aspects of delivering the CPP with its cargo into a vertebratesubject are also provided. Further aspects of CPPs and their deliveryare described in U.S. Pat. Nos. 8,575,305; 8,614,194 and 8,044,019. CPPscan be used to deliver the CRISPR-Cas system or components thereof. ThatCPPs can be employed to deliver the CRISPR-Cas system or componentsthereof is also provided in the manuscript “Gene disruption bycell-penetrating peptide-mediated delivery of Cas9 protein and guideRNA”, by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, etal. Genome Res. 2014 Apr. 2. [Epub ahead of print], incorporated byreference in its entirety, wherein it is demonstrated that treatmentwith CPP-conjugated recombinant Cas9 protein and CPP-complexed guideRNAs lead to endogenous gene disruptions in human cell lines. In thepaper the Cas9 protein was conjugated to CPP via a thioether bond,whereas the guide RNA was complexed with CPP, forming condensed,positively charged particles. It was shown that simultaneous andsequential treatment of human cells, including embryonic stem cells,dermal fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinomacells, with the modified Cas9 and guide RNA led to efficient genedisruptions with reduced off-target mutations relative to plasmidtransfections.

Implantable Devices

In another embodiment, implantable devices are also contemplated fordelivery of the nucleic acid-targeting system or component(s) thereof ornucleic acid molecule(s) coding therefor. For example, US PatentPublication 20110195123 discloses an implantable medical device whichelutes a drug locally and in prolonged period is provided, includingseveral types of such a device, the treatment modes of implementationand methods of implantation. The device comprising of polymericsubstrate, such as a matrix for example, that is used as the devicebody, and drugs, and in some cases additional scaffolding materials,such as metals or additional polymers, and materials to enhancevisibility and imaging. An implantable delivery device can beadvantageous in providing release locally and over a prolonged period,where drug is released directly to the extracellular matrix (ECM) of thediseased area such as tumor, inflammation, degeneration or forsymptomatic objectives, or to injured smooth muscle cells, or forprevention. One kind of drug is RNA, as disclosed above, and this systemmay be used and/or adapted to the nucleic acid-targeting system of thepresent invention. The modes of implantation in some embodiments areexisting implantation procedures that are developed and used today forother treatments, including brachytherapy and needle biopsy. In suchcases the dimensions of the new implant described in this invention aresimilar to the original implant. Typically a few devices are implantedduring the same treatment procedure.

US Patent Publication 20110195123, provides a drug delivery implantableor insertable system, including systems applicable to a cavity such asthe abdominal cavity and/or any other type of administration in whichthe drug delivery system is not anchored or attached, comprising abiostable and/or degradable and/or bioabsorbable polymeric substrate,which may for example optionally be a matrix. It should be noted thatthe term “insertion” also includes implantation. The drug deliverysystem is preferably implemented as a “Loder” as described in US PatentPublication 20110195123.

The polymer or plurality of polymers are biocompatible, incorporating anagent and/or plurality of agents, enabling the release of agent at acontrolled rate, wherein the total volume of the polymeric substrate,such as a matrix for example, in some embodiments is optionally andpreferably no greater than a maximum volume that permits a therapeuticlevel of the agent to be reached. As a non-limiting example, such avolume is preferably within the range of 0.1 m³ to 1000 mm³, as requiredby the volume for the agent load. The Loder may optionally be larger,for example when incorporated with a device whose size is determined byfunctionality, for example and without limitation, a knee joint, anintra-uterine or cervical ring and the like.

The drug delivery system (for delivering the composition) is designed insome embodiments to preferably employ degradable polymers, wherein themain release mechanism is bulk erosion; or in some embodiments, nondegradable, or slowly degraded polymers are used, wherein the mainrelease mechanism is diffusion rather than bulk erosion, so that theouter part functions as membrane, and its internal part functions as adrug reservoir, which practically is not affected by the surroundingsfor an extended period (for example from about a week to about a fewmonths). Combinations of different polymers with different releasemechanisms may also optionally be used. The concentration gradient atthe surface is preferably maintained effectively constant during asignificant period of the total drug releasing period, and therefore thediffusion rate is effectively constant (termed “zero mode” diffusion).By the term “constant” it is meant a diffusion rate that is preferablymaintained above the lower threshold of therapeutic effectiveness, butwhich may still optionally feature an initial burst and/or mayfluctuate, for example increasing and decreasing to a certain degree.The diffusion rate is preferably so maintained for a prolonged period,and it can be considered constant to a certain level to optimize thetherapeutically effective period, for example the effective silencingperiod.

The drug delivery system optionally and preferably is designed to shieldthe nucleotide based therapeutic agent from degradation, whetherchemical in nature or due to attack from enzymes and other factors inthe body of the subject.

The drug delivery system of US Patent Publication 20110195123 isoptionally associated with sensing and/or activation appliances that areoperated at and/or after implantation of the device, by non and/orminimally invasive methods of activation and/oracceleration/deceleration, for example optionally including but notlimited to thermal heating and cooling, laser beams, and ultrasonic,including focused ultrasound and/or RF (radiofrequency) methods ordevices.

According to some embodiments of US Patent Publication 20110195123, thesite for local delivery may optionally include target sitescharacterized by high abnormal proliferation of cells, and suppressedapoptosis, including tumors, active and/or chronic inflammation andinfection including autoimmune diseases states, degenerating tissueincluding muscle and nervous tissue, chronic pain, degenerative sites,and location of bone fractures and other wound locations for enhancementof regeneration of tissue, and injured cardiac, smooth and striatedmuscle.

The site for implantation of the composition, or target site, preferablyfeatures a radius, area and/or volume that is sufficiently small fortargeted local delivery. For example, the target site optionally has adiameter in a range of from about 0.1 mm to about 5 cm.

The location of the target site is preferably selected for maximumtherapeutic efficacy. For example, the composition of the drug deliverysystem (optionally with a device for implantation as described above) isoptionally and preferably implanted within or in the proximity of atumor environment, or the blood supply associated thereof.

For example the composition (optionally with the device) is optionallyimplanted within or in the proximity to pancreas, prostate, breast,liver, via the nipple, within the vascular system and so forth.

The target location is optionally selected from the group comprising,consisting essentially of, or consisting of (as non-limiting examplesonly, as optionally any site within the body may be suitable forimplanting a Loder): 1. brain at degenerative sites like in Parkinson orAlzheimer disease at the basal ganglia, white and gray matter; 2. spineas in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervixto prevent HPV infection; 4. active and chronic inflammatory joints; 5.dermis as in the case of psoriasis; 6. sympathetic and sensoric nervoussites for analgesic effect; 7. Intra osseous implantation; 8. acute andchronic infection sites; 9. Intra vaginal; 10. Inner ear-auditorysystem, labyrinth of the inner ear, vestibular system; 11. Intratracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder;14. biliary system; 15. parenchymal tissue including and not limited tothe kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18.dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21.Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominalcavity (for example but without limitation, for ovary cancer); 24. Intraesophageal and 25. Intra rectal.

Optionally insertion of the system (for example a device containing thecomposition) is associated with injection of material to the ECM at thetarget site and the vicinity of that site to affect local pH and/ortemperature and/or other biological factors affecting the diffusion ofthe drug and/or drug kinetics in the ECM, of the target site and thevicinity of such a site.

Optionally, according to some embodiments, the release of said agentcould be associated with sensing and/or activation appliances that areoperated prior and/or at and/or after insertion, by non and/or minimallyinvasive and/or else methods of activation and/oracceleration/deceleration, including laser beam, radiation, thermalheating and cooling, and ultrasonic, including focused ultrasound and/orRF (radiofrequency) methods or devices, and chemical activators.

According to other embodiments of US Patent Publication 20110195123, thedrug preferably comprises a RNA, for example for localized cancer casesin breast, pancreas, brain, kidney, bladder, lung, and prostate asdescribed below. Although exemplified with RNAi, many drugs areapplicable to be encapsulated in Loder, and can be used in associationwith this invention, as long as such drugs can be encapsulated with theLoder substrate, such as a matrix for example, and this system may beused and/or adapted to deliver the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, neuro and musculardegenerative diseases develop due to abnormal gene expression. Localdelivery of RNAs may have therapeutic properties for interfering withsuch abnormal gene expression. Local delivery of anti apoptotic, antiinflammatory and anti degenerative drugs including small drugs andmacromolecules may also optionally be therapeutic. In such cases theLoder is applied for prolonged release at constant rate and/or through adedicated device that is implanted separately. All of this may be usedand/or adapted to the nucleic acid-targeting system of the presentinvention.

As yet another example of a specific application, psychiatric andcognitive disorders are treated with gene modifiers. Gene knockdown is atreatment option. Loders locally delivering agents to central nervoussystem sites are therapeutic options for psychiatric and cognitivedisorders including but not limited to psychosis, bi-polar diseases,neurotic disorders and behavioral maladies. The Loders could alsodeliver locally drugs including small drugs and macromolecules uponimplantation at specific brain sites. All of this may be used and/oradapted to the nucleic acid-targeting system of the present invention.

As another example of a specific application, silencing of innate and/oradaptive immune mediators at local sites enables the prevention of organtransplant rejection. Local delivery of RNAs and immunomodulatingreagents with the Loder implanted into the transplanted organ and/or theimplanted site renders local immune suppression by repelling immunecells such as CD8 activated against the transplanted organ. All of thismay be used and/or adapted to the nucleic acid-targeting system of thepresent invention.

As another example of a specific application, vascular growth factorsincluding VEGFs and angiogenin and others are essential forneovascularization. Local delivery of the factors, peptides,peptidomimetics, or suppressing their repressors is an importanttherapeutic modality; silencing the repressors and local delivery of thefactors, peptides, macromolecules and small drugs stimulatingangiogenesis with the Loder is therapeutic for peripheral, systemic andcardiac vascular disease.

The method of insertion, such as implantation, may optionally already beused for other types of tissue implantation and/or for insertions and/orfor sampling tissues, optionally without modifications, or alternativelyoptionally only with non-major modifications in such methods. Suchmethods optionally include but are not limited to brachytherapy methods,biopsy, endoscopy with and/or without ultrasound, such as ERCP,stereotactic methods into the brain tissue, Laparoscopy, includingimplantation with a laparoscope into joints, abdominal organs, thebladder wall and body cavities.

Implantable device technology herein discussed can be employed withherein teachings and hence by this disclosure and the knowledge in theart, CRISPR-Cas system or components thereof or nucleic acid moleculesthereof or encoding or providing components may be delivered via animplantable device.

Patient-Specific Screening Methods

A nucleic acid-targeting system that targets RNA, e.g., trinucleotiderepeats can be used to screen patients or patent samples for thepresence of such repeats. The repeats can be the target of the RNA ofthe nucleic acid-targeting system, and if there is binding thereto bythe nucleic acid-targeting system, that binding can be detected, tothereby indicate that such a repeat is present. Thus, a nucleicacid-targeting system can be used to screen patients or patient samplesfor the presence of the repeat. The patient can then be administeredsuitable compound(s) to address the condition; or, can be administered anucleic acid-targeting system to bind to and cause insertion, deletionor mutation and alleviate the condition.

The invention uses nucleic acids to bind target RNA sequences.

CRISPR Effector Protein mRNA and Guide RNA

CRISPR effector protein mRNA and guide RNA might also be deliveredseparately. CRISPR effector protein mRNA can be delivered prior to theguide RNA to give time for CRISPR effector protein to be expressed.CRISPR effector protein mRNA might be administered 1-12 hours(preferably around 2-6 hours) prior to the administration of guide RNA.

Alternatively, CRISPR effector protein mRNA and guide RNA can beadministered together. Advantageously, a second booster dose of guideRNA can be administered 1-12 hours (preferably around 2-6 hours) afterthe initial administration of CRISPR effector protein mRNA+guide RNA.

The CRISPR effector protein of the present invention, i.e. a C2c2effector protein is sometimes referred to herein as a CRISPR Enzyme. Itwill be appreciated that the effector protein is based on or derivedfrom an enzyme, so the term ‘effector protein’ certainly includes‘enzyme’ in some embodiments. However, it will also be appreciated thatthe effector protein may, as required in some embodiments, have DNA orRNA binding, but not necessarily cutting or nicking, activity, includinga dead-Cas effector protein function.

Additional administrations of CRISPR effector protein mRNA and/or guideRNA might be useful to achieve the most efficient levels of genomemodification. In some embodiments, phenotypic alteration is preferablythe result of genome modification when a genetic disease is targeted,especially in methods of therapy and preferably where a repair templateis provided to correct or alter the phenotype.

In some embodiments diseases that may be targeted include thoseconcerned with disease-causing splice defects.

In some embodiments, cellular targets include HemopoieticStem/Progenitor Cells (CD34+); Human T cells; and Eye (retinalcells)—for example photoreceptor precursor cells.

In some embodiments Gene targets include: Human Beta Globin—HBB (fortreating Sickle Cell Anemia, including by stimulating gene-conversion(using closely related HBD gene as an endogenous template)); CD3(T-Cells); and CEP920—retina (eye).

In some embodiments disease targets also include: cancer; Sickle CellAnemia (based on a point mutation); HIV; Beta-Thalassemia; andophthalmic or ocular disease—for example Leber Congenital Amaurosis(LCA)-causing Splice Defect.

In some embodiments delivery methods include: Cationic Lipid Mediated“direct” delivery of Enzyme-Guide complex (ribonucleoprotein) andelectroporation of plasmid DNA.

Inventive methods can further comprise delivery of templates, such asrepair templates, which may be dsODN or ssODN, see below. Delivery oftemplates may be via the cotemporaneous or separate from delivery of anyor all the CRISPR effector protein or guide and via the same deliverymechanism or different. In some embodiments, it is preferred that thetemplate is delivered together with the guide, and, preferably, also theCRISPR effector protein. An example may be an AAV vector.

Inventive methods can further comprise: (a) delivering to the cell adouble-stranded oligodeoxynucleotide (dsODN) comprising overhangscomplimentary to the overhangs created by said double strand break,wherein said dsODN is integrated into the locus of interest; or—(b)delivering to the cell a single-stranded oligodeoxynucleotide (ssODN),wherein said ssODN acts as a template for homology directed repair ofsaid double strand break. Inventive methods can be for the prevention ortreatment of disease in an individual, optionally wherein said diseaseis caused by a defect in said locus of interest. Inventive methods canbe conducted in vivo in the individual or ex vivo on a cell taken fromthe individual, optionally wherein said cell is returned to theindividual.

For minimization of toxicity and off-target effect, it will be importantto control the concentration of CRISPR effector protein mRNA and guideRNA delivered. Optimal concentrations of CRISPR effector protein mRNAand guide RNA can be determined by testing different concentrations in acellular or animal model and using deep sequencing the analyze theextent of modification at potential off-target genomic loci. Forexample, for the guide sequence targeting 5′-GAGTCCGAGCAGAAGAAGAA-3′(SEQ ID NO: 53) in the EMX1 gene of the human genome, deep sequencingcan be used to assess the level of modification at the following twooff-target loci, 1: 5′-GAGTCCTAGCAGGAGAAGAA-3′ (SEQ ID NO: 54) and 2:5′-GAGTCTAAGCAGAAGAAGAA-3′ (SEQ ID NO: 55). The concentration that givesthe highest level of on-target modification while minimizing the levelof off-target modification should be chosen for in vivo delivery.

Inducible Systems

In some embodiments, a CRISPR effector protein may form a component ofan inducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc.), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR effector protein may be a part of a Light InducibleTranscriptional Effector (LITE) to direct changes in transcriptionalactivity in a sequence-specific manner. The components of a light mayinclude a CRISPR effector protein, a light-responsive cytochromeheterodimer (e.g. from Arabidopsis thaliana), and a transcriptionalactivation/repression domain. Further examples of inducible DNA bindingproteins and methods for their use are provided in U.S. 61/736,465 andU.S. 61/721,283, and WO 2014018423 A2 which is hereby incorporated byreference in its entirety.

Exemplary Methods of Using of CRISPR Cas System

The invention provides a non-naturally occurring or engineeredcomposition, or one or more polynucleotides encoding components of saidcomposition, or vector or delivery systems comprising one or morepolynucleotides encoding components of said composition for use in amodifying a target cell in vivo, ex vivo or in vitro and, may beconducted in a manner alters the cell such that once modified theprogeny or cell line of the CRISPR modified cell retains the alteredphenotype. The modified cells and progeny may be part of amulti-cellular organism such as a plant or animal with ex vivo or invivo application of CRISPR system to desired cell types. The CRISPRinvention may be a therapeutic method of treatment. The therapeuticmethod of treatment may comprise gene or genome editing, or genetherapy.

Modifying a Target with CRISPR Cas System or Complex (e.g., C2c2-RNAComplex)

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or invitro. In some embodiments, the method comprises sampling a cell orpopulation of cells from a human or non-human animal, and modifying thecell or cells. Culturing may occur at any stage ex vivo. The cell orcells may even be re-introduced into the non-human animal or plant. Forre-introduced cells it is particularly preferred that the cells are stemcells.

In some embodiments, the method comprises allowing a CRISPR complex tobind to the target polynucleotide to effect cleavage of said targetpolynucleotide thereby modifying the target polynucleotide, wherein theCRISPR complex comprises a CRISPR effector protein complexed with aguide sequence hybridized or hybridizable to a target sequence withinsaid target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a CRISPR complex to bind to the polynucleotidesuch that said binding results in increased or decreased expression ofsaid polynucleotide; wherein the CRISPR complex comprises a CRISPReffector protein complexed with a guide sequence hybridized orhybridizable to a target sequence within said polynucleotide. Similarconsiderations and conditions apply as above for methods of modifying atarget polynucleotide. In fact, these sampling, culturing andre-introduction options apply across the aspects of the presentinvention.

Indeed, in any aspect of the invention, the CRISPR complex may comprisea CRISPR effector protein complexed with a guide sequence hybridized orhybridizable to a target sequence. Similar considerations and conditionsapply as above for methods of modifying a target polynucleotide.

Thus in any of the non-naturally-occurring CRISPR effector proteinsdescribed herein comprise at least one modification and whereby theeffector protein has certain improved capabilities. In particular, anyof the effector proteins are capable of forming a CRISPR complex with aguide RNA. When such a complex forms, the guide RNA is capable ofbinding to a target polynucleotide sequence and the effector protein iscapable of modifying a target locus. In addition, the effector proteinin the CRISPR complex has reduced capability of modifying one or moreoff-target loci as compared to an unmodified enzyme/effector protein.

In addition, the modified CRISPR enzymes described herein encompassenzymes whereby in the CRISPR complex the effector protein has increasedcapability of modifying the one or more target loci as compared to anunmodified enzyme/effector protein. Such function may be providedseparate to or provided in combination with the above-described functionof reduced capability of modifying one or more off-target loci. Any sucheffector proteins may be provided with any of the further modificationsto the CRISPR effector protein as described herein, such as incombination with any activity provided by one or more associatedheterologous functional domains, any further mutations to reducenuclease activity and the like.

In advantageous embodiments of the invention, the modified CRISPReffector protein is provided with reduced capability of modifying one ormore off-target loci as compared to an unmodified enzyme/effectorprotein and increased capability of modifying the one or more targetloci as compared to an unmodified enzyme/effector protein. Incombination with further modifications to the effector protein,significantly enhanced specificity may be achieved. For example,combination of such advantageous embodiments with one or more additionalmutations is provided wherein the one or more additional mutations arein one or more catalytically active domains. In such effector proteins,enhanced specificity may be achieved due to an improved specificity interms of effector protein activity.

Additional functionalities which may be engineered into modified CRISPReffector proteins as described herein include the following. 1. ModifiedCRISPR effector proteins that disrupt RNA:protein interactions withoutaffecting protein tertiary or secondary structure. This includesresidues that contact any part of the RNA:RNA duplex. 2. Modified CRISPReffector proteins that weaken intra-protein interactions holding C2c2 inconformation essential for nuclease cutting in response to RNA binding(on or off target). For example: a modification that mildly inhibits,but still allows, the nuclease conformation of the HNH domain(positioned at the scissile phosphate). 3. Modified CRISPR effectorproteins that strengthen intra-protein interactions holding C2c2 in aconformation inhibiting nuclease activity in response to RNA binding (onor off targets). For example: a modification that stabilizes the HNHdomain in a conformation away from the scissile phosphate. Any suchadditional functional enhancement may be provided in combination withany other modification to the CRISPR effector protein as described indetail elsewhere herein.

Any of the herein described improved functionalities may be made to anyCRISPR effector protein, such as a C2c2 effector protein. However, itwill be appreciated that any of the functionalities described herein maybe engineered into C2c2 effector proteins from other orthologs,including chimeric effector proteins comprising fragments from multipleorthologs.

The invention uses nucleic acids to bind target DNA sequences. This isadvantageous as nucleic acids are much easier and cheaper to producethan proteins, and the specificity can be varied according to the lengthof the stretch where homology is sought. Complex 3-D positioning ofmultiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid”and “oligonucleotide” are used interchangeably. They refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Polynucleotides may have anythree dimensional structure, and may perform any function, known orunknown. The following are non-limiting examples of polynucleotides:coding or non-coding regions of a gene or gene fragment, loci (locus)defined from linkage analysis, exons, introns, messenger RNA (mRNA),transfer RNA, ribosomal RNA, short interfering RNA (siRNA),short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line. As used herein the term“variant” should be taken to mean the exhibition of qualities that havea pattern that deviates from what occurs in nature. The terms“non-naturally occurring” or “engineered” are used interchangeably andindicate the involvement of the hand of man. The terms, when referringto nucleic acid molecules or polypeptides mean that the nucleic acidmolecule or the polypeptide is at least substantially free from at leastone other component with which they are naturally associated in natureand as found in nature. “Complementarity” refers to the ability of anucleic acid to form hydrogen bond(s) with another nucleic acid sequenceby either traditional Watson-Crick base pairing or other non-traditionaltypes. A percent complementarity indicates the percentage of residues ina nucleic acid molecule which can form hydrogen bonds (e.g.,Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5,6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary). “Perfectly complementary” means that all the contiguousresidues of a nucleic acid sequence will hydrogen bond with the samenumber of contiguous residues in a second nucleic acid sequence.“Substantially complementary” as used herein refers to a degree ofcomplementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or morenucleotides, or refers to two nucleic acids that hybridize understringent conditions. As used herein, “stringent conditions” forhybridization refer to conditions under which a nucleic acid havingcomplementarity to a target sequence predominantly hybridizes with thetarget sequence, and substantially does not hybridize to non-targetsequences. Stringent conditions are generally sequence-dependent, andvary depending on a number of factors. In general, the longer thesequence, the higher the temperature at which the sequence specificallyhybridizes to its target sequence. Non-limiting examples of stringentconditions are described in detail in Tijssen (1993), LaboratoryTechniques In Biochemistry And Molecular Biology-Hybridization WithNucleic Acid Probes Part I, Second Chapter “Overview of principles ofhybridization and the strategy of nucleic acid probe assay”, Elsevier,N.Y. Where reference is made to a polynucleotide sequence, thencomplementary or partially complementary sequences are also envisaged.These are preferably capable of hybridizing to the reference sequenceunder highly stringent conditions. Generally, in order to maximize thehybridization rate, relatively low-stringency hybridization conditionsare selected: about 20 to 25° C. lower than the thermal melting point(T_(m)). The T_(m) is the temperature at which 50% of specific targetsequence hybridizes to a perfectly complementary probe in solution at adefined ionic strength and pH. Generally, in order to require at leastabout 85% nucleotide complementarity of hybridized sequences, highlystringent washing conditions are selected to be about 5 to 15° C. lowerthan the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30° C. lower than the T_(m).Highly permissive (very low stringency) washing conditions may be as lowas 500 C below the T_(m), allowing a high level of mis-matching betweenhybridized sequences. Those skilled in the art will recognize that otherphysical and chemical parameters in the hybridization and wash stagescan also be altered to affect the outcome of a detectable hybridizationsignal from a specific level of homology between target and probesequences. Preferred highly stringent conditions comprise incubation in50% formamide, 5×SSC, and 1% SDS at 42° C., or incubation in 5×SSC and1% SDS at 65° C., with wash in 0.2×SSC and 0.1% SDS at 65° C.“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogsteen binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the initiation of PCR, or the cleavage of apolynucleotide by an enzyme. A sequence capable of hybridizing with agiven sequence is referred to as the “complement” of the given sequence.As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions. As used herein, “expressionof a genomic locus” or “gene expression” is the process by whichinformation from a gene is used in the synthesis of a functional geneproduct. The products of gene expression are often proteins, but innon-protein coding genes such as rRNA genes or tRNA genes, the productis functional RNA. The process of gene expression is used by all knownlife—eukaryotes (including multicellular organisms), prokaryotes(bacteria and archaea) and viruses to generate functional products tosurvive. As used herein “expression” of a gene or nucleic acidencompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell. The terms “polypeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component. As used herein the term “aminoacid” includes natural and/or unnatural or synthetic amino acids,including glycine and both the D or L optical isomers, and amino acidanalogs and peptidomimetics. As used herein, the term “domain” or“protein domain” refers to a part of a protein sequence that may existand function independently of the rest of the protein chain. Asdescribed in aspects of the invention, sequence identity is related tosequence homology. Homology comparisons may be conducted by eye, or moreusually, with the aid of readily available sequence comparison programs.These commercially available computer programs may calculate percent (%)homology between two or more sequences and may also calculate thesequence identity shared by two or more amino acid or nucleic acidsequences.

In aspects of the invention the term “guide RNA”, refers to thepolynucleotide sequence comprising one or more of a putative oridentified tracr sequence and a putative or identified crRNA sequence orguide sequence. In particular embodiments, the “guide RNA” comprises aputative or identified crRNA sequence or guide sequence. In furtherembodiments, the guide RNA does not comprise a putative or identifiedtracr sequence.

As used herein the term “wild type” is a term of the art understood byskilled persons and means the typical form of an organism, strain, geneor characteristic as it occurs in nature as distinguished from mutant orvariant forms. A “wild type” can be a base line.

As used herein the term “variant” should be taken to mean the exhibitionof qualities that have a pattern that deviates from what occurs innature.

The terms “non-naturally occurring” or “engineered” are usedinterchangeably and indicate the involvement of the hand of man. Theterms, when referring to nucleic acid molecules or polypeptides meanthat the nucleic acid molecule or the polypeptide is at leastsubstantially free from at least one other component with which they arenaturally associated in nature and as found in nature. In all aspectsand embodiments, whether they include these terms or not, it will beunderstood that, preferably, the may be optional and thus preferablyincluded or not preferably not included. Furthermore, the terms“non-naturally occurring” and “engineered” may be used interchangeablyand so can therefore be used alone or in combination and one or othermay replace mention of both together. In particular, “engineered” ispreferred in place of “non-naturally occurring” or “non-naturallyoccurring and/or engineered.”

Sequence homologies may be generated by any of a number of computerprograms known in the art, for example BLAST or FASTA, etc. A suitablecomputer program for carrying out such an alignment is the GCG WisconsinBestfit package (University of Wisconsin, U.S.A.; Devereux et al., 1984,Nucleic Acids Research 12:387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 ibid—Chapter 18), FASTA (Atschul etal., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparisontools. Both BLAST and FASTA are available for offline and onlinesearching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). Howeverit is preferred to use the GCG Bestfit program. Percentage (%) sequencehomology may be calculated over contiguous sequences, i.e., one sequenceis aligned with the other sequence and each amino acid or nucleotide inone sequence is directly compared with the corresponding amino acid ornucleotide in the other sequence, one residue at a time. This is calledan “ungapped” alignment. Typically, such ungapped alignments areperformed only over a relatively short number of residues. Although thisis a very simple and consistent method, it fails to take intoconsideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion may cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without unduly penalizing the overall homology or identityscore. This is achieved by inserting “gaps” in the sequence alignment totry to maximize local homology or identity. However, these more complexmethods assign “gap penalties” to each gap that occurs in the alignmentso that, for the same number of identical amino acids, a sequencealignment with as few gaps as possible—reflecting higher relatednessbetween the two compared sequences—may achieve a higher score than onewith many gaps. “Affinity gap costs” are typically used that charge arelatively high cost for the existence of a gap and a smaller penaltyfor each subsequent residue in the gap. This is the most commonly usedgap scoring system. High gap penalties may, of course, produce optimizedalignments with fewer gaps. Most alignment programs allow the gappenalties to be modified. However, it is preferred to use the defaultvalues when using such software for sequence comparisons. For example,when using the GCG Wisconsin Bestfit package the default gap penalty foramino acid sequences is −12 for a gap and −4 for each extension.Calculation of maximum % homology therefore first requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984Nuc. Acids Research 12 p. 387). Examples of other software than mayperform sequence comparisons include, but are not limited to, the BLASTpackage (see Ausubel et al., 1999 Short Protocols in Molecular Biology,4^(th) Ed. —Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol.403-410) and the GENEWORKS suite of comparison tools. Both BLAST andFASTA are available for offline and online searching (see Ausubel etal., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60).However, for some applications, it is preferred to use the GCG Bestfitprogram. A new tool, called BLAST 2 Sequences is also available forcomparing protein and nucleotide sequences (see FEMS Microbiol Lett.1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and thewebsite of the National Center for Biotechnology information at thewebsite of the National Institutes for Health). Although the final %homology may be measured in terms of identity, the alignment processitself is typically not based on an all-or-nothing pair comparison.Instead, a scaled similarity score matrix is generally used that assignsscores to each pair-wise comparison based on chemical similarity orevolutionary distance. An example of such a matrix commonly used is theBLOSUM62 matrix—the default matrix for the BLAST suite of programs. GCGWisconsin programs generally use either the public default values or acustom symbol comparison table, if supplied (see user manual for furtherdetails). For some applications, it is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62. Alternatively, percentagehomologies may be calculated using the multiple alignment feature inDNASIS™ (Hitachi Software), based on an algorithm, analogous to CLUSTAL(Higgins D G & Sharp P M (1988), Gene 73(1), 237-244). Once the softwarehas produced an optimal alignment, it is possible to calculate %homology, preferably % sequence identity. The software typically doesthis as part of the sequence comparison and generates a numericalresult. The sequences may also have deletions, insertions orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent substance. Deliberate amino acidsubstitutions may be made on the basis of similarity in amino acidproperties (such as polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues) and it istherefore useful to group amino acids together in functional groups.Amino acids may be grouped together based on the properties of theirside chains alone. However, it is more useful to include mutation dataas well. The sets of amino acids thus derived are likely to be conservedfor structural reasons. These sets may be described in the form of aVenn diagram (Livingstone C. D. and Barton G. J. (1993) “Proteinsequence alignments: a strategy for the hierarchical analysis of residueconservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W. R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119;205-218). Conservative substitutions may be made, for example accordingto the table below which describes a generally accepted Venn diagramgrouping of amino acids.

Set Sub-set Hydrophobic F W Y H K M I L V A G C Aromatic F W Y HAliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E DPositively charged H K R Negatively charged E D Small V C A G S P T N DTiny A G S

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein to refer to a vertebrate, preferably a mammal,more preferably a human. Mammals include, but are not limited to,murines, simians, humans, farm animals, sport animals, and pets.Tissues, cells and their progeny of a biological entity obtained in vivoor cultured in vitro are also encompassed.

The terms “therapeutic agent”, “therapeutic capable agent” or “treatmentagent” are used interchangeably and refer to a molecule or compound thatconfers some beneficial effect upon administration to a subject. Thebeneficial effect includes enablement of diagnostic determinations;amelioration of a disease, symptom, disorder, or pathological condition;reducing or preventing the onset of a disease, symptom, disorder orcondition; and generally counteracting a disease, symptom, disorder orpathological condition.

As used herein, “treatment” or “treating,” or “palliating” or“ameliorating” are used interchangeably. These terms refer to anapproach for obtaining beneficial or desired results including but notlimited to a therapeutic benefit and/or a prophylactic benefit. Bytherapeutic benefit is meant any therapeutically relevant improvement inor effect on one or more diseases, conditions, or symptoms undertreatment. For prophylactic benefit, the compositions may beadministered to a subject at risk of developing a particular disease,condition, or symptom, or to a subject reporting one or more of thephysiological symptoms of a disease, even though the disease, condition,or symptom may not have yet been manifested.

The term “effective amount” or “therapeutically effective amount” refersto the amount of an agent that is sufficient to effect beneficial ordesired results. The therapeutically effective amount may vary dependingupon one or more of: the subject and disease condition being treated,the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art. The term also appliesto a dose that will provide an image for detection by any one of theimaging methods described herein. The specific dose may vary dependingon one or more of: the particular agent chosen, the dosing regimen to befollowed, whether it is administered in combination with othercompounds, timing of administration, the tissue to be imaged, and thephysical delivery system in which it is carried.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of immunology, biochemistry,chemistry, molecular biology, microbiology, cell biology, genomics andrecombinant DNA, which are within the skill of the art. See Sambrook,Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2ndedition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel,et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press,Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, ALABORATORY MANUAL, and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

Several aspects of the invention relate to vector systems comprising oneor more vectors, or vectors as such. Vectors can be designed forexpression of CRISPR transcripts (e.g. nucleic acid transcripts,proteins, or enzymes) in prokaryotic or eukaryotic cells. For example,CRISPR transcripts can be expressed in bacterial cells such asEscherichia coli, insect cells (using baculovirus expression vectors),yeast cells, or mammalian cells. Suitable host cells are discussedfurther in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, therecombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Embodiments of the invention include sequences (both polynucleotide orpolypeptide) which may comprise homologous substitution (substitutionand replacement are both used herein to mean the interchange of anexisting amino acid residue or nucleotide, with an alternative residueor nucleotide) that may occur i.e., like-for-like substitution in thecase of amino acids such as basic for basic, acidic for acidic, polarfor polar, etc. Non-homologous substitution may also occur i.e., fromone class of residue to another or alternatively involving the inclusionof unnatural amino acids such as ornithine (hereinafter referred to asZ), diaminobutyric acid ornithine (hereinafter referred to as B),norleucine ornithine (hereinafter referred to as O), pyridylalanine,thienylalanine, naphthylalanine and phenylglycine. Variant amino acidsequences may include suitable spacer groups that may be insertedbetween any two amino acid residues of the sequence including alkylgroups such as methyl, ethyl or propyl groups in addition to amino acidspacers such as glycine or P-alanine residues. A further form ofvariation, which involves the presence of one or more amino acidresidues in peptoid form, may be well understood by those skilled in theart. For the avoidance of doubt, “the peptoid form” is used to refer tovariant amino acid residues wherein the a-carbon substituent group is onthe residue's nitrogen atom rather than the α-carbon. Processes forpreparing peptides in the peptoid form are known in the art, for exampleSimon R J et al., PNAS (1992) 89(20), 9367-9371 and Horwell D C, TrendsBiotechnol. (1995) 13(4), 132-134.

Homology modelling: Corresponding residues in other C2c2 orthologs canbe identified by the methods of Zhang et al., 2012 (Nature; 490(7421):556-60) and Chen et al., 2015 (PLoS Comput Biol; 11(5): e1004248)—acomputational protein-protein interaction (PPI) method to predictinteractions mediated by domain-motif interfaces. PrePPI (PredictingPPI), a structure based PPI prediction method, combines structuralevidence with non-structural evidence using a Bayesian statisticalframework. The method involves taking a pair of query proteins and usingstructural alignment to identify structural representatives thatcorrespond to either their experimentally determined structures orhomology models. Structural alignment is further used to identify bothclose and remote structural neighbors by considering global and localgeometric relationships. Whenever two neighbors of the structuralrepresentatives form a complex reported in the Protein Data Bank, thisdefines a template for modelling the interaction between the two queryproteins. Models of the complex are created by superimposing therepresentative structures on their corresponding structural neighbor inthe template. This approach is further described in Dey et al., 2013(Prot Sci; 22: 359-66).

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as AmpliTaq Gold™, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR.

In certain aspects the invention involves vectors. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entityfrom one environment to another. It is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. Generally, avector is capable of replication when associated with the proper controlelements. In general, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. Vectors include, but are not limited to, nucleic acidmolecules that are single-stranded, double-stranded, or partiallydouble-stranded; nucleic acid molecules that comprise one or more freeends, no free ends (e.g., circular); nucleic acid molecules thatcomprise DNA, RNA, or both; and other varieties of polynucleotides knownin the art. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe inserted, such as by standard molecular cloning techniques. Anothertype of vector is a viral vector, wherein virally-derived DNA or RNAsequences are present in the vector for packaging into a virus (e.g.,retroviruses, replication defective retroviruses, adenoviruses,replication defective adenoviruses, and adeno-associated viruses(AAVs)). Viral vectors also include polynucleotides carried by a virusfor transfection into a host cell. Certain vectors are capable ofautonomous replication in a host cell into which they are introduced(e.g., bacterial vectors having a bacterial origin of replication andepisomal mammalian vectors). Other vectors (e.g., non-episomal mammalianvectors) are integrated into the genome of a host cell upon introductioninto the host cell, and thereby are replicated along with the hostgenome. Moreover, certain vectors are capable of directing theexpression of genes to which they are operatively-linked. Such vectorsare referred to herein as “expression vectors.” Common expressionvectors of utility in recombinant DNA techniques are often in the formof plasmids.

Recombinant expression vectors can comprise a nucleic acid of theinvention in a form suitable for expression of the nucleic acid in ahost cell, which means that the recombinant expression vectors includeone or more regulatory elements, which may be selected on the basis ofthe host cells to be used for expression, that is operatively-linked tothe nucleic acid sequence to be expressed. Within a recombinantexpression vector, “operably linked” is intended to mean that thenucleotide sequence of interest is linked to the regulatory element(s)in a manner that allows for expression of the nucleotide sequence (e.g.,in an in vitro transcription/translation system or in a host cell whenthe vector is introduced into the host cell). With regards torecombination and cloning methods, mention is made of U.S. patentapplication Ser. No. 10/815,730, published Sep. 2, 2004 as US2004-0171156 A1, the contents of which are herein incorporated byreference in their entirety.

Aspects of the invention relate to bicistronic vectors for guide RNA andwild type, modified or mutated CRISPR effector proteins/enzymes (e.g.C2c2). Bicistronic expression vectors guide RNA and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) are preferred.In general and particularly in this embodiment and wild type, modifiedor mutated CRISPR effector proteins/enzymes (e.g. C2c2) is preferablydriven by the CBh promoter. The RNA may preferably be driven by a PolIII promoter, such as a U6 promoter. Ideally the two are combined.

In some embodiments, a loop in the guide RNA is provided. This may be astem loop or a tetra loop. The loop is preferably GAAA, but it is notlimited to this sequence or indeed to being only 4 bp in length. Indeed,preferred loop forming sequences for use in hairpin structures are fournucleotides in length, and most preferably have the sequence GAAA.However, longer or shorter loop sequences may be used, as mayalternative sequences. The sequences preferably include a nucleotidetriplet (for example, AAA), and an additional nucleotide (for example Cor G). Examples of loop forming sequences include CAAA and AAAG.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The term “regulatory element” is intended to include promoters,enhancers, internal ribosomal entry sites (IRES), and other expressioncontrol elements (e.g., transcription termination signals, such aspolyadenylation signals and poly-U sequences). Such regulatory elementsare described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).Regulatory elements include those that direct constitutive expression ofa nucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). A tissue-specific promoter maydirect expression primarily in a desired tissue of interest, such asmuscle, neuron, bone, skin, blood, specific organs (e.g., liver,pancreas), or particular cell types (e.g., lymphocytes). Regulatoryelements may also direct expression in a temporal-dependent manner, suchas in a cell-cycle dependent or developmental stage-dependent manner,which may or may not also be tissue or cell-type specific. In someembodiments, a vector comprises one or more pol III promoter (e.g., 1,2, 3, 4, 5, or more pol III promoters), one or more pol II promoters(e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol Ipromoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), orcombinations thereof. Examples of pol III promoters include, but are notlimited to, U6 and H1 promoters. Examples of pol II promoters include,but are not limited to, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al.,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers;the R-U 5′ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p.466-472, 1988); SV40 enhancer; and the intron sequence between exons 2and 3 of rabbit β-globin (Proc. Natl. Acad. Sci. U.S.A., Vol. 78(3), p.1527-31, 1981). It will be appreciated by those skilled in the art thatthe design of the expression vector can depend on such factors as thechoice of the host cell to be transformed, the level of expressiondesired, etc. A vector can be introduced into host cells to therebyproduce transcripts, proteins, or peptides, including fusion proteins orpeptides, encoded by nucleic acids as described herein (e.g., clusteredregularly interspersed short palindromic repeats (CRISPR) transcripts,proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).With regards to regulatory sequences, mention is made of U.S. patentapplication Ser. No. 10/491,026, the contents of which are incorporatedby reference herein in their entirety. With regards to promoters,mention is made of PCT publication WO 2011/028929 and U.S. applicationSer. No. 12/511,940, the contents of which are incorporated by referenceherein in their entirety.

Vectors can be designed for expression of CRISPR transcripts (e.g.,nucleic acid transcripts, proteins, or enzymes) in prokaryotic oreukaryotic cells. For example, CRISPR transcripts can be expressed inbacterial cells such as Escherichia coli, insect cells (usingbaculovirus expression vectors), yeast cells, or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Vectors may be introduced and propagated in a prokaryote or prokaryoticcell. In some embodiments, a prokaryote is used to amplify copies of avector to be introduced into a eukaryotic cell or as an intermediatevector in the production of a vector to be introduced into a eukaryoticcell (e.g., amplifying a plasmid as part of a viral vector packagingsystem). In some embodiments, a prokaryote is used to amplify copies ofa vector and express one or more nucleic acids, such as to provide asource of one or more proteins for delivery to a host cell or hostorganism. Expression of proteins in prokaryotes is most often carriedout in Escherichia coli with vectors containing constitutive orinducible promoters directing the expression of either fusion ornon-fusion proteins. Fusion vectors add a number of amino acids to aprotein encoded therein, such as to the amino terminus of therecombinant protein. Such fusion vectors may serve one or more purposes,such as: (i) to increase expression of recombinant protein; (ii) toincrease the solubility of the recombinant protein; and (iii) to aid inthe purification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Example fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET lid(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

In some embodiments, a vector is a yeast expression vector. Examples ofvectors for expression in yeast Saccharomyces cerevisiae includepYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan andHerskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), andpicZ (InVitrogen Corp, San Diego, Calif.).

In some embodiments, a vector drives protein expression in insect cellsusing baculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., SF9 cells)include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Luckow and Summers, 1989. Virology 170:31-39).

In some embodiments, a vector is capable of driving expression of one ormore sequences in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, 1987.Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).When used in mammalian cells, the expression vector's control functionsare typically provided by one or more regulatory elements. For example,commonly used promoters are derived from polyoma, adenovirus 2,cytomegalovirus, simian virus 40, and others disclosed herein and knownin the art. For other suitable expression systems for both prokaryoticand eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al.,MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989.

In some embodiments, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the a-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546). With regards to theseprokaryotic and eukaryotic vectors, mention is made of U.S. Pat. No.6,750,059, the contents of which are incorporated by reference herein intheir entirety. Other embodiments of the invention may relate to the useof viral vectors, with regards to which mention is made of U.S. patentapplication Ser. No. 13/092,085, the contents of which are incorporatedby reference herein in their entirety. Tissue-specific regulatoryelements are known in the art and in this regard, mention is made ofU.S. Pat. No. 7,776,321, the contents of which are incorporated byreference herein in their entirety.

In some embodiments, a regulatory element is operably linked to one ormore elements of a CRISPR system so as to drive expression of the one ormore elements of the CRISPR system. In general, CRISPRs (ClusteredRegularly Interspaced Short Palindromic Repeats), also known as SPIDRs(SPacer Interspersed Direct Repeats), constitute a family of DNA locithat are usually specific to a particular bacterial species. The CRISPRlocus comprises a distinct class of interspersed short sequence repeats(SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol.,169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553-3556[1989]), and associated genes. Similar interspersed SSRs have beenidentified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena,and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol.,10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999];Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica etal., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differfrom other SSRs by the structure of the repeats, which have been termedshort regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ.Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246[2000]). In general, the repeats are short elements that occur inclusters that are regularly spaced by unique intervening sequences witha substantially constant length (Mojica et al., [2000], supra). Althoughthe repeat sequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., J. Bacteriol.,182:2393-2401 [2000]). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575[2002]; and Mojica et al., [2005]) including, but not limited toAeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Haloarcula,Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus,Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium,Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus,Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma,Fusobacterium, Azoarcus, Chromobacterium, Neisseria, Nitrosomonas,Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella,Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus,Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia,Treponema, and Thermotoga.

In general, “nucleic acid-targeting system” as used in the presentapplication refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of nucleicacid-targeting CRISPR-associated (“Cas”) genes (also referred to hereinas an effector protein), including sequences encoding a nucleicacid-targeting Cas (effector) protein and a guide RNA (comprising crRNAsequence and a trans-activating CRISPR/Cas system RNA (tracrRNA)sequence), or other sequences and transcripts from a nucleicacid-targeting CRISPR locus. In some embodiments, one or more elementsof a nucleic acid-targeting system are derived from a Type V/Type VInucleic acid-targeting CRISPR system. In some embodiments, one or moreelements of a nucleic acid-targeting system is derived from a particularorganism comprising an endogenous nucleic acid-targeting CRISPR system.In general, a nucleic acid-targeting system is characterized by elementsthat promote the formation of a nucleic acid-targeting complex at thesite of a target sequence. In the context of formation of a nucleicacid-targeting complex, “target sequence” refers to a sequence to whicha guide sequence is designed to have complementarity, wherehybridization between a target sequence and a guide RNA promotes theformation of a DNA or RNA-targeting complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a nucleic acid-targetingcomplex. A target sequence may comprise RNA polynucleotides. In someembodiments, a target sequence is located in the nucleus or cytoplasm ofa cell. In some embodiments, the target sequence may be within anorganelle of a eukaryotic cell, for example, mitochondrion orchloroplast. A sequence or template that may be used for recombinationinto the targeted locus comprising the target sequences is referred toas an “editing template” or “editing RNA” or “editing sequence”. Inaspects of the invention, an exogenous template RNA may be referred toas an editing template. In an aspect of the invention the recombinationis homologous recombination.

Typically, in the context of an endogenous nucleic acid-targetingsystem, formation of a nucleic acid-targeting complex (comprising aguide RNA hybridized to a target sequence and complexed with one or morenucleic acid-targeting effector proteins) results in cleavage of one orboth RNA strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 50, or more base pairs from) the target sequence. In someembodiments, one or more vectors driving expression of one or moreelements of a nucleic acid-targeting system are introduced into a hostcell such that expression of the elements of the nucleic acid-targetingsystem direct formation of a nucleic acid-targeting complex at one ormore target sites. For example, a nucleic acid-targeting effectorprotein and a guide RNA could each be operably linked to separateregulatory elements on separate vectors. Alternatively, two or more ofthe elements expressed from the same or different regulatory elements,may be combined in a single vector, with one or more additional vectorsproviding any components of the nucleic acid-targeting system notincluded in the first vector. nucleic acid-targeting system elementsthat are combined in a single vector may be arranged in any suitableorientation, such as one element located 5′ with respect to (“upstream”of) or 3′ with respect to (“downstream” of) a second element. The codingsequence of one element may be located on the same or opposite strand ofthe coding sequence of a second element, and oriented in the same oropposite direction. In some embodiments, a single promoter drivesexpression of a transcript encoding a nucleic acid-targeting effectorprotein and a guide RNA embedded within one or more intron sequences(e.g. each in a different intron, two or more in at least one intron, orall in a single intron). In some embodiments, the nucleic acid-targetingeffector protein and guide RNA are operably linked to and expressed fromthe same promoter.

In general, a guide sequence is any polynucleotide sequence havingsufficient complementarity with a target polynucleotide sequence tohybridize with the target sequence and direct sequence-specific bindingof a nucleic acid-targeting complex to the target sequence. In someembodiments, the degree of complementarity between a guide sequence andits corresponding target sequence, when optimally aligned using asuitable alignment algorithm, is about or more than about 50%, 60%, 75%,80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may bedetermined with the use of any suitable algorithm for aligningsequences, non-limiting example of which include the Smith-Watermanalgorithm, the Needleman-Wunsch algorithm, algorithms based on theBurrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW,Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, SanDiego, Calif.)), SOAP (available at soap.genomics.org.cn), and Maq(available at maq.sourceforge.net). In some embodiments, a guidesequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75,or more nucleotides in length. In some embodiments, a guide sequence isless than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewernucleotides in length. The ability of a guide sequence to directsequence-specific binding of a nucleic acid-targeting complex to atarget sequence may be assessed by any suitable assay. For example, thecomponents of a nucleic acid-targeting system sufficient to form anucleic acid-targeting complex, including the guide sequence to betested, may be provided to a host cell having the corresponding targetsequence, such as by transfection with vectors encoding the componentsof the nucleic acid-targeting CRISPR sequence, followed by an assessmentof preferential cleavage within or in the vicinity of the targetsequence, such as by Surveyor assay as described herein. Similarly,cleavage of a target polynucleotide sequence (or a sequence in thevicinity thereof) may be evaluated in a test tube by providing thetarget sequence, components of a nucleic acid-targeting complex,including the guide sequence to be tested and a control guide sequencedifferent from the test guide sequence, and comparing binding or rate ofcleavage at or in the vicinity of the target sequence between the testand control guide sequence reactions. Other assays are possible, andwill occur to those skilled in the art.

A guide sequence may be selected to target any target sequence. In someembodiments, the target sequence is a sequence within a gene transcriptor mRNA.

In some embodiments, the target sequence is a sequence within a genomeof a cell.

In some embodiments, a guide sequence is selected to reduce the degreeof secondary structure within the guide sequence. Secondary structuremay be determined by any suitable polynucleotide folding algorithm. Someprograms are based on calculating the minimal Gibbs free energy. Anexample of one such algorithm is mFold, as described by Zuker andStiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example foldingalgorithm is the online webserver RNAfold, developed at Institute forTheoretical Chemistry at the University of Vienna, using the centroidstructure prediction algorithm (see e.g. A. R. Gruber et al., 2008, Cell106(1): 23-24; and P A Carr and G M Church, 2009, Nature Biotechnology27(12): 1151-62). Further algorithms may be found in WO 2014/093709,incorporated herein by reference.

In some embodiments, the nucleic acid-targeting effector protein is partof a fusion protein comprising one or more heterologous protein domains(e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or moredomains in addition to the nucleic acid-targeting effector protein). Insome embodiments, the CRISPR effector protein/enzyme is part of a fusionprotein comprising one or more heterologous protein domains (e.g. aboutor more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains inaddition to the CRISPR enzyme). A CRISPR effector protein/enzyme fusionprotein may comprise any additional protein sequence, and optionally alinker sequence between any two domains. Examples of protein domainsthat may be fused to an effector protein include, without limitation,epitope tags, reporter gene sequences, and protein domains having one ormore of the following activities: methylase activity, demethylaseactivity, transcription activation activity, transcription repressionactivity, transcription release factor activity, histone modificationactivity, RNA cleavage activity and nucleic acid binding activity.Non-limiting examples of epitope tags include histidine (His) tags, V5tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-Gtags, and thioredoxin (Trx) tags. Examples of reporter genes include,but are not limited to, glutathione-S-transferase (GST), horseradishperoxidase (HRP), chloramphenicol acetyltransferase (CAT)beta-galactosidase, beta-glucuronidase, luciferase, green fluorescentprotein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellowfluorescent protein (YFP), and autofluorescent proteins including bluefluorescent protein (BFP). A nucleic acid-targeting effector protein maybe fused to a gene sequence encoding a protein or a fragment of aprotein that bind DNA molecules or bind other cellular molecules,including but not limited to maltose binding protein (MBP), S-tag, Lex ADNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, andherpes simplex virus (HSV) VP16 protein fusions. Additional domains thatmay form part of a fusion protein comprising a nucleic acid-targetingeffector protein are described in US20110059502, incorporated herein byreference. In some embodiments, a tagged nucleic acid-targeting effectorprotein is used to identify the location of a target sequence.

In some embodiments, a CRISPR enzyme may form a component of aninducible system. The inducible nature of the system would allow forspatiotemporal control of gene editing or gene expression using a formof energy. The form of energy may include but is not limited toelectromagnetic radiation, sound energy, chemical energy and thermalenergy. Examples of inducible system include tetracycline induciblepromoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptionactivations systems (FKBP, ABA, etc.), or light inducible systems(Phytochrome, LOV domains, or cryptochrome). In one embodiment, theCRISPR enzyme may be a part of a Light Inducible TranscriptionalEffector (LITE) to direct changes in transcriptional activity in asequence-specific manner. The components of a light may include a CRISPRenzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsisthaliana), and a transcriptional activation/repression domain. Furtherexamples of inducible DNA binding proteins and methods for their use areprovided in U.S. 61/736,465 and U.S. 61/721,283 and WO 2014/018423 andU.S. Pat. Nos. 8,889,418, 8,895,308, US20140186919, US20140242700,US20140273234, US20140335620, WO2014093635, which is hereby incorporatedby reference in its entirety.

In some aspects, the invention provides methods comprising deliveringone or more polynucleotides, such as or one or more vectors as describedherein, one or more transcripts thereof, and/or one or proteinstranscribed therefrom, to a host cell. In some aspects, the inventionfurther provides cells produced by such methods, and organisms (such asanimals, plants, or fungi) comprising or produced from such cells. Insome embodiments, a nucleic acid-targeting effector protein incombination with (and optionally complexed with) a guide RNA isdelivered to a cell. Conventional viral and non-viral based genetransfer methods can be used to introduce nucleic acids in mammaliancells or target tissues. Such methods can be used to administer nucleicacids encoding components of a nucleic acid-targeting system to cells inculture, or in a host organism. Non-viral vector delivery systemsinclude DNA plasmids, RNA (e.g. a transcript of a vector describedherein), naked nucleic acid, and nucleic acid complexed with a deliveryvehicle, such as a liposome. Viral vector delivery systems include DNAand RNA viruses, which have either episomal or integrated genomes afterdelivery to the cell. For a review of gene therapy procedures, seeAnderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon,TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt,Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology andNeuroscience 8:35-36 (1995); Kremer & Perricaudet, British MedicalBulletin 51(1):31-44 (1995); Haddada et al., in Current Topics inMicrobiology and Immunology, Doerfler and Bohm (eds) (1995); and Yu etal., Gene Therapy 1:13-26 (1994).

Methods of non-viral delivery of nucleic acids include lipofection,nucleofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells(e.g. in vitro or ex vivo administration) or target tissues (e.g. invivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids takes advantage of highly evolved processes for targeting a virusto specific cells in the body and trafficking the viral payload to thenucleus. Viral vectors can be administered directly to patients (invivo) or they can be used to treat cells in vitro, and the modifiedcells may optionally be administered to patients (ex vivo). Conventionalviral based systems could include retroviral, lentivirus, adenoviral,adeno-associated and herpes simplex virus vectors for gene transfer.Integration in the host genome is possible with the retrovirus,lentivirus, and adeno-associated virus gene transfer methods, oftenresulting in long term expression of the inserted transgene.Additionally, high transduction efficiencies have been observed in manydifferent cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system would thereforedepend on the target tissue. Retroviral vectors are comprised ofcis-acting long terminal repeats with packaging capacity for up to 6-10kb of foreign sequence. The minimum cis-acting LTRs are sufficient forreplication and packaging of the vectors, which are then used tointegrate the therapeutic gene into the target cell to provide permanenttransgene expression. Widely used retroviral vectors include those basedupon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV),Simian Immunodeficiency virus (SIV), human Immunodeficiency virus (HIV),and combinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992);Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700). In applications where transient expression ispreferred, adenoviral based systems may be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand levels of expression have been obtained. This vector can be producedin large quantities in a relatively simple system. Adeno-associatedvirus (“AAV”) vectors may also be used to transduce cells with targetnucleic acids, e.g., in the in vitro production of nucleic acids andpeptides, and for in vivo and ex vivo gene therapy procedures (see,e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368;WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J.Clin. Invest. 94:1351 (1994)). Construction of recombinant AAV vectorsare described in a number of publications, including U.S. Pat. No.5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985);Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat &Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol.63:03822-3828 (1989).

Models of Genetic and Epigenetic Conditions

A method of the invention may be used to create a plant, an animal orcell that may be used to model and/or study genetic or epigeneticconditions of interest, such as a through a model of mutations ofinterest or a disease model. As used herein, “disease” refers to adisease, disorder, or indication in a subject. For example, a method ofthe invention may be used to create an animal or cell that comprises amodification in one or more nucleic acid sequences associated with adisease, or a plant, animal or cell in which the expression of one ormore nucleic acid sequences associated with a disease are altered. Sucha nucleic acid sequence may encode a disease associated protein sequenceor may be a disease associated control sequence. Accordingly, it isunderstood that in embodiments of the invention, a plant, subject,patient, organism or cell can be a non-human subject, patient, organismor cell. Thus, the invention provides a plant, animal or cell, producedby the present methods, or a progeny thereof. The progeny may be a cloneof the produced plant or animal, or may result from sexual reproductionby crossing with other individuals of the same species to introgressfurther desirable traits into their offspring. The cell may be in vivoor ex vivo in the cases of multicellular organisms, particularly animalsor plants. In the instance where the cell is in cultured, a cell linemay be established if appropriate culturing conditions are met andpreferably if the cell is suitably adapted for this purpose (forinstance a stem cell). Bacterial cell lines produced by the inventionare also envisaged. Hence, cell lines are also envisaged.

In some methods, the disease model can be used to study the effects ofmutations on the animal or cell and development and/or progression ofthe disease using measures commonly used in the study of the disease.Alternatively, such a disease model is useful for studying the effect ofa pharmaceutically active compound on the disease.

In some methods, the disease model can be used to assess the efficacy ofa potential gene therapy strategy. That is, a disease-associated gene orpolynucleotide can be modified such that the disease development and/orprogression is inhibited or reduced. In particular, the method comprisesmodifying a disease-associated gene or polynucleotide such that analtered protein is produced and, as a result, the animal or cell has analtered response. Accordingly, in some methods, a genetically modifiedanimal may be compared with an animal predisposed to development of thedisease such that the effect of the gene therapy event may be assessed.

In another embodiment, this invention provides a method of developing abiologically active agent that modulates a cell signaling eventassociated with a disease gene. The method comprises contacting a testcompound with a cell comprising one or more vectors that driveexpression of one or more of a CRISPR enzyme, and a direct repeatsequence linked to a guide sequence; and detecting a change in a readoutthat is indicative of a reduction or an augmentation of a cell signalingevent associated with, e.g., a mutation in a disease gene contained inthe cell.

A cell model or animal model can be constructed in combination with themethod of the invention for screening a cellular function change. Such amodel may be used to study the effects of a genome sequence modified bythe CRISPR complex of the invention on a cellular function of interest.For example, a cellular function model may be used to study the effectof a modified genome sequence on intracellular signaling orextracellular signaling. Alternatively, a cellular function model may beused to study the effects of a modified genome sequence on sensoryperception. In some such models, one or more genome sequences associatedwith a signaling biochemical pathway in the model are modified.

Several disease models have been specifically investigated. Theseinclude de novo autism risk genes CHD8, KATNAL2, and SCN2A; and thesyndromic autism (Angelman Syndrome) gene UBE3A. These genes andresulting autism models are of course preferred, but serve to show thebroad applicability of the invention across genes and correspondingmodels.

An altered expression of one or more genome sequences associated with asignalling biochemical pathway can be determined by assaying for adifference in the mRNA levels of the corresponding genes between thetest model cell and a control cell, when they are contacted with acandidate agent. Alternatively, the differential expression of thesequences associated with a signaling biochemical pathway is determinedby detecting a difference in the level of the encoded polypeptide orgene product.

To assay for an agent-induced alteration in the level of mRNAtranscripts or corresponding polynucleotides, nucleic acid contained ina sample is first extracted according to standard methods in the art.For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (1989), or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by the manufacturers. The mRNAcontained in the extracted nucleic acid sample is then detected byamplification procedures or conventional hybridization assays (e.g.Northern blot analysis) according to methods widely known in the art orbased on the methods exemplified herein.

For purpose of this invention, amplification means any method employinga primer and a polymerase capable of replicating a target sequence withreasonable fidelity. Amplification may be carried out by natural orrecombinant DNA polymerases such as AmpliTaq Gold™, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. Apreferred amplification method is PCR. In particular, the isolated RNAcan be subjected to a reverse transcription assay that is coupled with aquantitative polymerase chain reaction (RT-PCR) in order to quantify theexpression level of a sequence associated with a signaling biochemicalpathway.

Detection of the gene expression level can be conducted in real time inan amplification assay. In one aspect, the amplified products can bedirectly visualized with fluorescent DNA-binding agents including butnot limited to DNA intercalators and DNA groove binders. Because theamount of the intercalators incorporated into the double-stranded DNAmolecules is typically proportional to the amount of the amplified DNAproducts, one can conveniently determine the amount of the amplifiedproducts by quantifying the fluorescence of the intercalated dye usingconventional optical systems in the art. DNA-binding dye suitable forthis application include SYBR green, SYBR blue, DAPI, propidium iodine,Oeste, SYBR gold, ethidium bromide, acridines, proflavine, acridineorange, acriflavine, fluorocoumarin, ellipticine, daunomycin,chloroquine, distamycin D, chromomycin, homidium, mithramycin, rutheniumpolypyridyls, anthramycin, and the like.

In another aspect, other fluorescent labels such as sequence specificprobes can be employed in the amplification reaction to facilitate thedetection and quantification of the amplified products. Probe-basedquantitative amplification relies on the sequence-specific detection ofa desired amplified product. It utilizes fluorescent, target-specificprobes (e.g., TaqMan® probes) resulting in increased specificity andsensitivity. Methods for performing probe-based quantitativeamplification are well established in the art and are taught in U.S.Pat. No. 5,210,015.

In yet another aspect, conventional hybridization assays usinghybridization probes that share sequence homology with sequencesassociated with a signaling biochemical pathway can be performed.Typically, probes are allowed to form stable complexes with thesequences associated with a signaling biochemical pathway containedwithin the biological sample derived from the test subject in ahybridization reaction. It will be appreciated by one of skill in theart that where antisense is used as the probe nucleic acid, the targetpolynucleotides provided in the sample are chosen to be complementary tosequences of the antisense nucleic acids. Conversely, where thenucleotide probe is a sense nucleic acid, the target polynucleotide isselected to be complementary to sequences of the sense nucleic acid.

Hybridization can be performed under conditions of various stringency.Suitable hybridization conditions for the practice of the presentinvention are such that the recognition interaction between the probeand sequences associated with a signaling biochemical pathway is bothsufficiently specific and sufficiently stable. Conditions that increasethe stringency of a hybridization reaction are widely known andpublished in the art. See, for example, (Sambrook, et al., (1989);Nonradioactive In Situ Hybridization Application Manual, BoehringerMannheim, second edition). The hybridization assay can be formed usingprobes immobilized on any solid support, including but are not limitedto nitrocellulose, glass, silicon, and a variety of gene arrays. Apreferred hybridization assay is conducted on high-density gene chips asdescribed in U.S. Pat. No. 5,445,934.

For a convenient detection of the probe-target complexes formed duringthe hybridization assay, the nucleotide probes are conjugated to adetectable label. Detectable labels suitable for use in the presentinvention include any composition detectable by photochemical,biochemical, spectroscopic, immunochemical, electrical, optical orchemical means. A wide variety of appropriate detectable labels areknown in the art, which include fluorescent or chemiluminescent labels,radioactive isotope labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, B-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The detection methods used to detect or quantify the hybridizationintensity will typically depend upon the label selected above. Forexample, radiolabels may be detected using photographic film or aphosphoimager. Fluorescent markers may be detected and quantified usinga photodetector to detect emitted light. Enzymatic labels are typicallydetected by providing the enzyme with a substrate and measuring thereaction product produced by the action of the enzyme on the substrate;and finally colorimetric labels are detected by simply visualizing thecolored label.

An agent-induced change in expression of sequences associated with asignaling biochemical pathway can also be determined by examining thecorresponding gene products. Determining the protein level typicallyinvolves (a) contacting the protein contained in a biological samplewith an agent that specifically bind to a protein associated with asignaling biochemical pathway; and (b) identifying any agent:proteincomplex so formed. In one aspect of this embodiment, the agent thatspecifically binds a protein associated with a signaling biochemicalpathway is an antibody, preferably a monoclonal antibody.

The reaction is performed by contacting the agent with a sample of theproteins associated with a signaling biochemical pathway derived fromthe test samples under conditions that will allow a complex to formbetween the agent and the proteins associated with a signalingbiochemical pathway. The formation of the complex can be detecteddirectly or indirectly according to standard procedures in the art. Inthe direct detection method, the agents are supplied with a detectablelabel and unreacted agents may be removed from the complex; the amountof remaining label thereby indicating the amount of complex formed. Forsuch method, it is preferable to select labels that remain attached tothe agents even during stringent washing conditions. It is preferablethat the label does not interfere with the binding reaction. In thealternative, an indirect detection procedure may use an agent thatcontains a label introduced either chemically or enzymatically. Adesirable label generally does not interfere with binding or thestability of the resulting agent:polypeptide complex. However, the labelis typically designed to be accessible to an antibody for an effectivebinding and hence generating a detectable signal.

A wide variety of labels suitable for detecting protein levels are knownin the art. Non-limiting examples include radioisotopes, enzymes,colloidal metals, fluorescent compounds, bioluminescent compounds, andchemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the bindingreaction can be quantified by standard quantitative assays. Asillustrated above, the formation of agent:polypeptide complex can bemeasured directly by the amount of label remained at the site ofbinding. In an alternative, the protein associated with a signalingbiochemical pathway is tested for its ability to compete with a labeledanalog for binding sites on the specific agent. In this competitiveassay, the amount of label captured is inversely proportional to theamount of protein sequences associated with a signaling biochemicalpathway present in a test sample.

A number of techniques for protein analysis based on the generalprinciples outlined above are available in the art. They include but arenot limited to radioimmunoassays, ELISA (enzyme linked immunoradiometricassays), “sandwich” immunoassays, immunoradiometric assays, in situimmunoassays (using e.g., colloidal gold, enzyme or radioisotopelabels), western blot analysis, immunoprecipitation assays,immunofluorescent assays, and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associatedwith a signaling biochemical pathway are preferable for conducting theaforementioned protein analyses. Where desired, antibodies thatrecognize a specific type of post-translational modifications (e.g.,signaling biochemical pathway inducible modifications) can be used.Post-translational modifications include but are not limited toglycosylation, lipidation, acetylation, and phosphorylation. Theseantibodies may be purchased from commercial vendors. For example,anti-phosphotyrosine antibodies that specifically recognizetyrosine-phosphorylated proteins are available from a number of vendorsincluding Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodiesare particularly useful in detecting proteins that are differentiallyphosphorylated on their tyrosine residues in response to an ER stress.Such proteins include but are not limited to eukaryotic translationinitiation factor 2 alpha (eIF-2α). Alternatively, these antibodies canbe generated using conventional polyclonal or monoclonal antibodytechnologies by immunizing a host animal or an antibody-producing cellwith a target protein that exhibits the desired post-translationalmodification.

In practicing the subject method, it may be desirable to discern theexpression pattern of an protein associated with a signaling biochemicalpathway in different bodily tissue, in different cell types, and/or indifferent subcellular structures. These studies can be performed withthe use of tissue-specific, cell-specific or subcellular structurespecific antibodies capable of binding to protein markers that arepreferentially expressed in certain tissues, cell types, or subcellularstructures.

An altered expression of a gene associated with a signaling biochemicalpathway can also be determined by examining a change in activity of thegene product relative to a control cell. The assay for an agent-inducedchange in the activity of a protein associated with a signalingbiochemical pathway will dependent on the biological activity and/or thesignal transduction pathway that is under investigation. For example,where the protein is a kinase, a change in its ability to phosphorylatethe downstream substrate(s) can be determined by a variety of assaysknown in the art. Representative assays include but are not limited toimmunoblotting and immunoprecipitation with antibodies such asanti-phosphotyrosine antibodies that recognize phosphorylated proteins.In addition, kinase activity can be detected by high throughputchemiluminescent assays such as AlphaScreen™ (available from PerkinElmer) and eTag™ assay (Chan-Hui, et al. (2003) Clinical Immunology 111:162-174).

Where the protein associated with a signaling biochemical pathway ispart of a signaling cascade leading to a fluctuation of intracellular pHcondition, pH sensitive molecules such as fluorescent pH dyes can beused as the reporter molecules. In another example where the proteinassociated with a signaling biochemical pathway is an ion channel,fluctuations in membrane potential and/or intracellular ionconcentration can be monitored. A number of commercial kits andhigh-throughput devices are particularly suited for a rapid and robustscreening for modulators of ion channels. Representative instrumentsinclude FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences).These instruments are capable of detecting reactions in over 1000 samplewells of a microplate simultaneously, and providing real-timemeasurement and functional data within a second or even a minisecond.

In practicing any of the methods disclosed herein, a suitable vector canbe introduced to a cell or an embryo via one or more methods known inthe art, including without limitation, microinjection, electroporation,sonoporation, biolistics, calcium phosphate-mediated transfection,cationic transfection, liposome transfection, dendrimer transfection,heat shock transfection, nucleofection transfection, magnetofection,lipofection, impalefection, optical transfection, proprietaryagent-enhanced uptake of nucleic acids, and delivery via liposomes,immunoliposomes, virosomes, or artificial virions. In some methods, thevector is introduced into an embryo by microinjection. The vector orvectors may be microinjected into the nucleus or the cytoplasm of theembryo. In some methods, the vector or vectors may be introduced into acell by nucleofection.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA).

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

The target polynucleotide of a CRISPR complex can be any polynucleotideendogenous or exogenous to the eukaryotic cell. For example, the targetpolynucleotide can be a polynucleotide residing in the nucleus of theeukaryotic cell. The target polynucleotide can be a sequence coding agene product (e.g., a protein) or a non-coding sequence (e.g., aregulatory polynucleotide or a junk DNA). Without wishing to be bound bytheory, it is believed that the target sequence should be associatedwith a PAM (protospacer adjacent motif); that is, a short sequencerecognized by the CRISPR complex. The precise sequence and lengthrequirements for the PAM differ depending on the CRISPR enzyme used, butPAMs are typically 2-5 base pair sequences adjacent the protospacer(that is, the target sequence) Examples of PAM sequences are given inthe examples section below, and the skilled person will be able toidentify further PAM sequences for use with a given CRISPR enzyme.

The target polynucleotide of a CRISPR complex may include a number ofdisease associated genes and polynucleotides as well as signalingbiochemical pathway-associated genes and polynucleotides as listed inU.S. provisional patent applications 61/736,527 and 61/748,427 bothentitled SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATIONfiled on Dec. 12, 2012 and Jan. 2, 2013, respectively, and PCTApplication PCT/US2013/074667, entitled DELIVERY, ENGINEERING ANDOPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION AND THERAPEUTIC APPLICATIONS, filed Dec. 12, 2013, thecontents of all of which are herein incorporated by reference in theirentirety.

Examples of target polynucleotides include a sequence associated with asignaling biochemical pathway, e.g., a signaling biochemicalpathway-associated gene or polynucleotide. Examples of targetpolynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene orpolynucleotide which is yielding transcription or translation productsat an abnormal level or in an abnormal form in cells derived from adisease-affected tissues compared with tissues or cells of a non diseasecontrol. It may be a gene that becomes expressed at an abnormally highlevel; it may be a gene that becomes expressed at an abnormally lowlevel, where the altered expression correlates with the occurrenceand/or progression of the disease. A disease-associated gene also refersto a gene possessing mutation(s) or genetic variation that is directlyresponsible or is in linkage disequilibrium with a gene(s) that isresponsible for the etiology of a disease. The transcribed or translatedproducts may be known or unknown, and may be at a normal or abnormallevel.

Transcriptome Wide Knock-down Screening

The CRISPR effector protein complexes described herein can be used toperform efficient and cost effective functional transcriptomic screens.Such screens can utilize CRISPR effector protein based transcriptomewide libraries. Such screens and libraries can provide for determiningthe function of genes, cellular pathways genes are involved in, and howany alteration in gene expression can result in a particular biologicalprocess. An advantage of the present invention is that the CRISPR systemavoids off-target binding and its resulting side effects. This isachieved using systems arranged to have a high degree of sequencespecificity for the target DNA. In preferred embodiments of theinvention, the CRISPR effector protein complexes are C2c2 effectorprotein complexes.

In embodiments of the invention, a transcriptome wide library maycomprise a plurality of C2c2 guide RNAs, as described herein, comprisingguide sequences that are capable of targeting a plurality of targetsequences in a plurality of loci in a population of eukaryotic cells.The population of cells may be a population of embryonic stem (ES)cells. The target sequence in the locus may be a non-coding sequence.The non-coding sequence may be an intron, regulatory sequence, splicesite, 3′ UTR, 5′ UTR, or polyadenylation signal. Gene function of one ormore gene products may be altered by said targeting. The targeting mayresult in a knockout of gene function. The targeting of a gene productmay comprise more than one guide RNA. A gene product may be targeted by2, 3, 4, 5, 6, 7, 8, 9, or 10 guide RNAs, preferably 3 to 4 per gene.Off-target modifications may be minimized by exploiting the staggereddouble strand breaks generated by C2c2 effector protein complexes or byutilizing methods analogous to those used in CRISPR-Cas9 systems (See,e.g., DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,Scott, D., Weinstein, J., Ran, FA., Konermann, S., Agarwala, V., Li, Y.,Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L A., Bao, G.,& Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013)), incorporatedherein by reference. The targeting may be of about 100 or moresequences. The targeting may be of about 1000 or more sequences. Thetargeting may be of about 20,000 or more sequences. The targeting may beof the entire genome. The targeting may be of a panel of targetsequences focused on a relevant or desirable pathway. The pathway may bean immune pathway. The pathway may be a cell division pathway.

One aspect of the invention comprehends a transcriptome wide librarythat may comprise a plurality of C2c2 guide RNAs that may comprise guidesequences that are capable of targeting a plurality of target sequencesin a plurality of loci, wherein said targeting results in a knockdown ofgene function. This library may potentially comprise guide RNAs thattarget each and every gene in the genome of an organism.

In some embodiments of the invention the organism or subject is aeukaryote (including mammal including human) or a non-human eukaryote ora non-human animal or a non-human mammal. In some embodiments, theorganism or subject is a non-human animal, and may be an arthropod, forexample, an insect, or may be a nematode. In some methods of theinvention the organism or subject is a plant. In some methods of theinvention the organism or subject is a mammal or a non-human mammal. Anon-human mammal may be for example a rodent (preferably a mouse or arat), an ungulate, or a primate. In some methods of the invention theorganism or subject is algae, including microalgae, or is a fungus.

The knockdown of gene function may comprise: introducing into each cellin the population of cells a vector system of one or more vectorscomprising an engineered, non-naturally occurring C2c2 effector proteinsystem comprising I. a C2c2 effector protein, and II. one or more guideRNAs, wherein components I and II may be same or on different vectors ofthe system, integrating components I and II into each cell, wherein theguide sequence targets a unique gene in each cell, wherein the C2c2effector protein is operably linked to a regulatory element, whereinwhen transcribed, the guide RNA comprising the guide sequence directssequence-specific binding of the C2c2 effector protein system to atarget sequence in the genomic loci of the unique gene, inducingcleavage of the genomic loci by the C2c2 effector protein, andconfirming different knockdown events in a plurality of unique genes ineach cell of the population of cells thereby generating a gene knockdowncell library. The invention comprehends that the population of cells isa population of eukaryotic cells, and in a preferred embodiment, thepopulation of cells is a population of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be asingle vector comprising a C2c2 effector protein, a sgRNA, andoptionally, a selection marker into target cells. Not being bound by atheory, the ability to simultaneously deliver a C2c2 effector proteinand sgRNA through a single vector enables application to any cell typeof interest, without the need to first generate cell lines that expressthe C2c2 effector protein. The regulatory element may be an induciblepromoter. The inducible promoter may be a doxycycline induciblepromoter. In some methods of the invention the expression of the guidesequence is under the control of the T7 promoter and is driven by theexpression of T7 polymerase. The confirming of different knockdownevents may be by whole transcriptome sequencing. The knockdown event maybe achieved in 100 or more unique genes. The knockdown event may beachieved in 1000 or more unique genes. The knockdown event may beachieved in 20,000 or more unique genes. The knockdown event may beachieved in the entire transcriptome. The knockdown of gene function maybe achieved in a plurality of unique genes which function in aparticular physiological pathway or condition. The pathway or conditionmay be an immune pathway or condition. The pathway or condition may be acell division pathway or condition.

The invention also provides kits that comprise the transcriptome widelibraries mentioned herein. The kit may comprise a single containercomprising vectors or plasmids comprising the library of the invention.The kit may also comprise a panel comprising a selection of unique C2c2effector protein system guide RNAs comprising guide sequences from thelibrary of the invention, wherein the selection is indicative of aparticular physiological condition. The invention comprehends that thetargeting is of about 100 or more sequences, about 1000 or moresequences or about 20,000 or more sequences or the entire transcriptome.Furthermore, a panel of target sequences may be focused on a relevant ordesirable pathway, such as an immune pathway or cell division.

In an additional aspect of the invention, the C2c2 effector protein maycomprise one or more mutations and may be used as a generic RNA bindingprotein with or without fusion to a functional domain. The mutations maybe artificially introduced mutations or gain- or loss-of-functionmutations. The mutations have been characterized as described herein. Inone aspect of the invention, the functional domain may be atranscriptional activation domain, which may be VP64. In other aspectsof the invention, the functional domain may be a transcriptionalrepressor domain, which may be KRAB or SID4X. Other aspects of theinvention relate to the mutated C2c2 effector protein being fused todomains which include but are not limited to a transcriptionalactivator, repressor, a recombinase, a transposase, a histone remodeler,a demethylase, a DNA methyltransferase, a cryptochrome, a lightinducible/controllable domain or a chemically inducible/controllabledomain. Some methods of the invention can include inducing expression oftargeted genes. In one embodiment, inducing expression by targeting aplurality of target sequences in a plurality of genomic loci in apopulation of eukaryotic cells is by use of a functional domain.

Useful in the practice of the instant invention utilizing C2c2 effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to:

Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O.,Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson, T.,Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F. ScienceDecember 12. (2013). [Epub ahead of print]; Published in final editedform as: Science. 2014 Jan. 3; 343(6166): 84-87.

Shalem et al. involves a new way to interrogate gene function on agenome-wide scale. Their studies showed that delivery of a genome-scaleCRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751unique guide sequences enabled both negative and positive selectionscreening in human cells. First, the authors showed use of the GeCKOlibrary to identify genes essential for cell viability in cancer andpluripotent stem cells. Next, in a melanoma model, the authors screenedfor genes whose loss is involved in resistance to vemurafenib, atherapeutic that inhibits mutant protein kinase BRAF. Their studiesshowed that the highest-ranking candidates included previously validatedgenes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1.The authors observed a high level of consistency between independentguide RNAs targeting the same gene and a high rate of hit confirmation,and thus demonstrated the promise of genome-scale screening with Cas9.

Reference is also made to US patent publication number US20140357530;and PCT Patent Publication WO2014093701, hereby incorporated herein byreference.

Functional Alteration and Screening

In another aspect, the present invention provides for a method offunctional evaluation and screening of genes. The use of the CRISPRsystem of the present invention to precisely deliver functional domains,to activate or repress genes or to alter epigenetic state by preciselyaltering the methylation site on a specific locus of interest, can bewith one or more guide RNAs applied to a single cell or population ofcells or with a library applied to genome in a pool of cells ex vivo orin vivo comprising the administration or expression of a librarycomprising a plurality of guide RNAs (sgRNAs) and wherein the screeningfurther comprises use of a C2c2 effector protein, wherein the CRISPRcomplex comprising the C2c2 effector protein is modified to comprise aheterologous functional domain. In an aspect the invention provides amethod for screening a genome/transcriptome comprising theadministration to a host or expression in a host in vivo of a library.In an aspect the invention provides a method as herein discussed furthercomprising an activator administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussedwherein the activator is attached to a C2c2 effector protein. In anaspect the invention provides a method as herein discussed wherein theactivator is attached to the N terminus or the C terminus of the C2c2effector protein. In an aspect the invention provides a method as hereindiscussed wherein the activator is attached to a sgRNA loop. In anaspect the invention provides a method as herein discussed furthercomprising a repressor administered to the host or expressed in thehost. In an aspect the invention provides a method as herein discussed,wherein the screening comprises affecting and detecting gene activation,gene inhibition, or cleavage in the locus.

In an aspect, the invention provides efficient on-target activity andminimizes off target activity. In an aspect, the invention providesefficient on-target cleavage by C2c2 effector protein and minimizesoff-target cleavage by the C2c2 effector protein. In an aspect, theinvention provides guide specific binding of C2c2 effector protein at agene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides target-specific gene regulation. In an aspect, theinvention provides guide specific binding of C2c2 effector protein at agene locus without DNA cleavage. Accordingly, in an aspect, theinvention provides for cleavage at one locus and gene regulation at adifferent locus using a single C2c2 effector protein. In an aspect, theinvention provides orthogonal activation and/or inhibition and/orcleavage of multiple targets using one or more C2c2 effector proteinand/or enzyme.

In an aspect the invention provides a method as herein discussed,wherein the host is a eukaryotic cell. In an aspect the inventionprovides a method as herein discussed, wherein the host is a mammaliancell. In an aspect the invention provides a method as herein discussed,wherein the host is a non-human eukaryote. In an aspect the inventionprovides a method as herein discussed, wherein the non-human eukaryoteis a non-human mammal. In an aspect the invention provides a method asherein discussed, wherein the non-human mammal is a mouse. An aspect theinvention provides a method as herein discussed comprising the deliveryof the C2c2 effector protein complexes or component(s) thereof ornucleic acid molecule(s) coding therefor, wherein said nucleic acidmolecule(s) are operatively linked to regulatory sequence(s) andexpressed in vivo. In an aspect the invention provides a method asherein discussed wherein the expressing in vivo is via a lentivirus, anadenovirus, or an AAV. In an aspect the invention provides a method asherein discussed wherein the delivery is via a particle, a nanoparticle,a lipid or a cell penetrating peptide (CPP).

In an aspect the invention provides a pair of CRISPR complexescomprising C2c2 effector protein, each comprising a guide RNA (sgRNA)comprising a guide sequence capable of hybridizing to a target sequencein a genomic locus of interest in a cell, wherein at least one loop ofeach sgRNA is modified by the insertion of distinct RNA sequence(s) thatbind to one or more adaptor proteins, and wherein the adaptor protein isassociated with one or more functional domains, wherein each sgRNA ofeach C2c2 effector protein complex comprises a functional domain havinga DNA cleavage activity.

In an aspect the invention provides a method for cutting a targetsequence in a locus of interest comprising delivery to a cell of theC2c2 effector protein complexes or component(s) thereof or nucleic acidmolecule(s) coding therefor, wherein said nucleic acid molecule(s) areoperatively linked to regulatory sequence(s) and expressed in vivo. Inan aspect the invention provides a method as herein-discussed whereinthe delivery is via a lentivirus, an adenovirus, or an AAV.

In an aspect the invention provides a library, method or complex asherein-discussed wherein the sgRNA is modified to have at least onenon-coding functional loop, e.g., wherein the at least one non-codingfunctional loop is repressive; for instance, wherein the at least onenon-coding functional loop comprises Alu.

In one aspect, the invention provides a method for altering or modifyingexpression of a gene product. The said method may comprise introducinginto a cell containing and expressing a DNA molecule encoding the geneproduct an engineered, non-naturally occurring CRISPR system comprisinga C2c2 effector protein and guide RNA that targets the RNA molecule,whereby the guide RNA targets the RNA target molecule encoding the geneproduct and the C2c2 effector protein cleaves the RNA molecule encodingthe gene product, whereby expression of the gene product is altered;and, wherein the C2c2 effector protein and the guide RNA do notnaturally occur together. The invention comprehends the guide RNAcomprising a guide sequence linked to a direct repeat sequence. Theinvention further comprehends the C2c2 effector protein being codonoptimized for expression in a Eukaryotic cell. In a preferred embodimentthe Eukaryotic cell is a mammalian cell and in a more preferredembodiment the mammalian cell is a human cell. In a further embodimentof the invention, the expression of the gene product is decreased.

In some embodiments, one or more functional domains are associated withthe C2c2 effector protein. In some embodiments, one or more functionaldomains are associated with an adaptor protein, for example as used withthe modified guides of Konnerman et al. (Nature 517, 583-588, 29 Jan.2015). In some embodiments, one or more functional domains areassociated with an dead sgRNA (dRNA). In some embodiments, a dRNAcomplex with active C2c2 effector protein directs gene regulation by afunctional domain at on gene locus while an sgRNA directs DNA cleavageby the active C2c2 effector protein at another locus, for example asdescribed analogously in CRISPR-Cas9 systems by Dahlman et al.,‘Orthogonal gene control with a catalytically active Cas9 nuclease,’Nature Biotechnology 33, p. 1159-61 (November, 2015). In someembodiments, dRNAs are selected to maximize selectivity of regulationfor a gene locus of interest compared to off-target regulation. In someembodiments, dRNAs are selected to maximize target gene regulation andminimize target cleavage.

For the purposes of the following discussion, reference to a functionaldomain could be a functional domain associated with the C2c2 effectorprotein or a functional domain associated with the adaptor protein.

In some embodiments, the one or more functional domains is an NLS(Nuclear Localization Sequence) or an NES (Nuclear Export Signal). Insome embodiments, the one or more functional domains is atranscriptional activation domain comprises VP64, p65, MyoD1, HSF1, RTA,SET7/9 and a histone acetyltransferase. Other references herein toactivation (or activator) domains in respect of those associated withthe CRISPR enzyme include any known transcriptional activation domainand specifically VP64, p65, MyoD1, HSF1, RTA, SET7/9 or a histoneacetyltransferase.

In some embodiments, the one or more functional domains is atranscriptional repressor domain. In some embodiments, thetranscriptional repressor domain is a KRAB domain. In some embodiments,the transcriptional repressor domain is a NUE domain, NcoR domain, SIDdomain or a SID4X domain.

In some embodiments, the one or more functional domains have one or moreactivities comprising translation activation activity, translationrepression activity, methylase activity, demethylase activity,transcription activation activity, transcription repression activity,transcription release factor activity, histone modification activity,RNA cleavage activity, DNA cleavage activity, DNA integration activityor nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments.Exemplary histone modifying domains are discussed below. Transposasedomains, HR (Homologous Recombination) machinery domains, recombinasedomains, and/or integrase domains are also preferred as the presentfunctional domains. In some embodiments, DNA integration activityincludes HR machinery domains, integrase domains, recombinase domainsand/or transposase domains. Histone acetyltransferases are preferred insome embodiments.

In some embodiments, the DNA cleavage activity is due to a nuclease. Insome embodiments, the nuclease comprises a Fok1 nuclease. See, “DimericCRISPR RNA-guided FokI nucleases for highly specific genome editing”,Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden,Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J.Keith Joung Nature Biotechnology 32(6): 569-77 (2014), relates todimeric RNA-guided FokI Nucleases that recognize extended sequences andcan edit endogenous genes with high efficiencies in human cells.

In some embodiments, the one or more functional domains is attached tothe C2c2 effector protein so that upon binding to the sgRNA and targetthe functional domain is in a spatial orientation allowing for thefunctional domain to function in its attributed function.

In some embodiments, the one or more functional domains is attached tothe adaptor protein so that upon binding of the C2c2 effector protein tothe sgRNA and target, the functional domain is in a spatial orientationallowing for the functional domain to function in its attributedfunction.

In an aspect the invention provides a composition as herein discussedwherein the one or more functional domains is attached to the C2c2effector protein or adaptor protein via a linker, optionally a Gly-Serlinker, as discussed herein.

It is also preferred to target endogenous (regulatory) control elements,such as involved in translation, stability, etc. Targeting of knowncontrol elements can be used to activate or repress the gene ofinterest. Targeting of putative control elements on the other hand canbe used as a means to verify such elements (by measuring the translationof the gene of interest) or to detect novel control elements. Inaddition, targeting of putative control elements can be useful in thecontext of understanding genetic causes of disease. Many mutations andcommon SNP variants associated with disease phenotypes are locatedoutside coding regions. Targeting of such regions with either theactivation or repression systems described herein can be followed byreadout of transcription of either a) a set of putative targets (e.g. aset of genes located in closest proximity to the control element) or b)whole-transcriptome readout by e.g. RNA-seq or microarray. This wouldallow for the identification of likely candidate genes involved in thedisease phenotype. Such candidate genes could be useful as novel drugtargets.

Histone acetyltransferase (HAT) inhibitors are mentioned herein.However, an alternative in some embodiments is for the one or morefunctional domains to comprise an acetyltransferase, preferably ahistone acetyltransferase. These are useful in the field of epigenomics,for example in methods of interrogating the epigenome. Methods ofinterrogating the epigenome may include, for example, targetingepigenomic sequences. Targeting epigenomic sequences may include theguide being directed to an epigenomic target sequence. Epigenomic targetsequence may include, in some embodiments, include a promoter, silenceror an enhancer sequence.

Use of a functional domain linked to a C2c2 effector protein asdescribed herein, preferably a dead-C2c2 effector protein, morepreferably a dead-FnC2c2 effector protein, to target epigenomicsequences can be used to activate or repress promoters, silencer orenhancers.

Examples of acetyltransferases are known but may include, in someembodiments, histone acetyltransferases. In some embodiments, thehistone acetyltransferase may comprise the catalytic core of the humanacetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6th April2015).

In some preferred embodiments, the functional domain is linked to adead-C2c2 effector protein to target and activate epigenomic sequencessuch as promoters or enhancers. One or more guides directed to suchpromoters or enhancers may also be provided to direct the binding of theCRISPR enzyme to such promoters or enhancers.

In certain embodiments, the RNA targeting effector protein of theinvention can be used to interfere with co-transcriptional modificationsof DNA/chromatin structure, RNA-directed DNA methylation, orRNA-directed silencing/activation of DNA/chromatin. RNA-directed DNAmethylation (RdDM) is an epigenetic process first discovered in plants.During RdDM, double-stranded RNAs (dsRNAs) are processed to 21-24nucleotide small interfering RNAs (siRNAs) and guide methylation ofhomologous DNA loci. Besides RNA molecules, a plethora of proteins areinvolved in the establishment of RdDM, like Argonautes, DNAmethyltransferases, chromatin remodelling complexes and theplant-specific PolIV and PolV. All these act in concert to add amethyl-group at the 5′ position of cytosines. Small RNAs can modify thechromatin structure and silence transcription by guidingArgonaute-containing complexes to complementary nascent (non-coding) RNAtranscripts. Subsequently the recruitment of chromatin-modifyingcomplexes, including histone and DNA methyltransferases, is mediated.The RNA targeting effector protein of the invention may be used totarget such small RNAs and interfere in interactions between these smallRNAs and the nascent non-coding transcripts.

The term “associated with” is used here in relation to the associationof the functional domain to the C2c2 effector protein or the adaptorprotein. It is used in respect of how one molecule ‘associates’ withrespect to another, for example between an adaptor protein and afunctional domain, or between the C2c2 effector protein and a functionaldomain. In the case of such protein-protein interactions, thisassociation may be viewed in terms of recognition in the way an antibodyrecognizes an epitope. Alternatively, one protein may be associated withanother protein via a fusion of the two, for instance one subunit beingfused to another subunit. Fusion typically occurs by addition of theamino acid sequence of one to that of the other, for instance viasplicing together of the nucleotide sequences that encode each proteinor subunit. Alternatively, this may essentially be viewed as bindingbetween two molecules or direct linkage, such as a fusion protein. Inany event, the fusion protein may include a linker between the twosubunits of interest (i.e. between the enzyme and the functional domainor between the adaptor protein and the functional domain). Thus, in someembodiments, the C2c2 effector protein or adaptor protein is associatedwith a functional domain by binding thereto. In other embodiments, theC2c2 effector protein or adaptor protein is associated with a functionaldomain because the two are fused together, optionally via anintermediate linker.

Saturating Mutagenesis

The C2c2 effector protein system(s) described herein can be used toperform saturating or deep scanning mutagenesis of genomic loci inconjunction with a cellular phenotype—for instance, for determiningcritical minimal features and discrete vulnerabilities of functionalelements required for gene expression, drug resistance, and reversal ofdisease. By saturating or deep scanning mutagenesis is meant that everyor essentially every RNA base is cut within the genomic loci. A libraryof C2c2 effector protein guide RNAs may be introduced into a populationof cells. The library may be introduced, such that each cell receives asingle guide RNA (sgRNA). In the case where the library is introduced bytransduction of a viral vector, as described herein, a low multiplicityof infection (MOI) is used. The library may include sgRNAs targetingevery sequence upstream of a (protospacer adjacent motif) (PAM) sequencein a genomic locus. The library may include at least 100 non-overlappinggenomic sequences upstream of a PAM sequence for every 1000 base pairswithin the genomic locus. The library may include sgRNAs targetingsequences upstream of at least one different PAM sequence. The C2c2effector protein systems may include more than one C2c2 protein. AnyC2c2 effector protein as described herein, including orthologues orengineered C2c2 effector proteins that recognize different PAM sequencesmay be used. The frequency of off target sites for a sgRNA may be lessthan 500. Off target scores may be generated to select sgRNAs with thelowest off target sites. Any phenotype determined to be associated withcutting at a sgRNA target site may be confirmed by using sgRNAstargeting the same site in a single experiment. Validation of a targetsite may also be performed by using a modified C2c2 effector protein, asdescribed herein, and two sgRNAs targeting the genomic site of interest.Not being bound by a theory, a target site is a true hit if the changein phenotype is observed in validation experiments.

The C2c2 effector protein system(s) for saturating or deep scanningmutagenesis can be used in a population of cells. The C2c2 effectorprotein system(s) can be used in eukaryotic cells, including but notlimited to mammalian and plant cells. The population of cells may beprokaryotic cells. The population of eukaryotic cells may be apopulation of embryonic stem (ES) cells, neuronal cells, epithelialcells, immune cells, endocrine cells, muscle cells, erythrocytes,lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention provides for a method of screeningfor functional elements associated with a change in a phenotype. Thelibrary may be introduced into a population of cells that are adapted tocontain a C2c2 effector protein. The cells may be sorted into at leasttwo groups based on the phenotype. The phenotype may be expression of agene, cell growth, or cell viability. The relative representation of theguide RNAs present in each group are determined, whereby genomic sitesassociated with the change in phenotype are determined by therepresentation of guide RNAs present in each group. The change inphenotype may be a change in expression of a gene of interest. The geneof interest may be upregulated, downregulated, or knocked out. The cellsmay be sorted into a high expression group and a low expression group.The population of cells may include a reporter construct that is used todetermine the phenotype. The reporter construct may include a detectablemarker. Cells may be sorted by use of the detectable marker.

In another aspect, the present invention provides for a method ofscreening for loci associated with resistance to a chemical compound.The chemical compound may be a drug or pesticide. The library may beintroduced into a population of cells that are adapted to contain a C2c2effector protein, wherein each cell of the population contains no morethan one guide RNA; the population of cells are treated with thechemical compound; and the representation of guide RNAs are determinedafter treatment with the chemical compound at a later time point ascompared to an early time point, whereby genomic sites associated withresistance to the chemical compound are determined by enrichment ofguide RNAs. Representation of sgRNAs may be determined by deepsequencing methods.

Useful in the practice of the instant invention utilizing C2c2 effectorprotein complexes are methods used in CRISPR-Cas9 systems and referenceis made to the article entitled BCL11A enhancer dissection byCas9-mediated in situ saturating mutagenesis. Canver, M. C., Smith, E.C., Sher, F., Pinello, L., Sanjana, N. E., Shalem, O., Chen, D. D.,Schupp, P. G., Vinjamur, D. S., Garcia, S. P., Luc, S., Kurita, R.,Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Orkin, S.H., & Bauer, D. E. DOI:10.1038/nature15521, published online Sep. 16,2015, the article is herein incorporated by reference and discussedbriefly below:

Canver et al. involves novel pooled CRISPR-Cas9 guide RNA libraries toperform in situ saturating mutagenesis of the human and mouse BCL11Aerythroid enhancers previously identified as an enhancer associated withfetal hemoglobin (HbF) level and whose mouse ortholog is necessary forerythroid BCL11A expression. This approach revealed critical minimalfeatures and discrete vulnerabilities of these enhancers. Throughediting of primary human progenitors and mouse transgenesis, the authorsvalidated the BCL11A erythroid enhancer as a target for HbF reinduction.The authors generated a detailed enhancer map that informs therapeuticgenome editing.

Method of Using C2c2 Systems to Modify a Cell or Organism

The invention in some embodiments comprehends a method of modifying acell or organism. The cell may be a prokaryotic cell or a eukaryoticcell. The cell may be a mammalian cell. The mammalian cell many be anon-human primate, bovine, porcine, rodent or mouse cell. The cell maybe a non-mammalian eukaryotic cell such as poultry, fish or shrimp. Thecell may also be a plant cell. The plant cell may be of a crop plantsuch as cassava, corn, sorghum, wheat, or rice. The plant cell may alsobe of an algae, tree or vegetable. The modification introduced to thecell by the present invention may be such that the cell and progeny ofthe cell are altered for improved production of biologic products suchas an antibody, starch, alcohol or other desired cellular output. Themodification introduced to the cell by the present invention may be suchthat the cell and progeny of the cell include an alteration that changesthe biologic product produced.

The system may comprise one or more different vectors. In an aspect ofthe invention, the effector protein is codon optimized for expressionthe desired cell type, preferentially a eukaryotic cell, preferably amammalian cell or a human cell.

Packaging cells are typically used to form virus particles that arecapable of infecting a host cell. Such cells include 293 cells, whichpackage adenovirus, and ψ2 cells or PA317 cells, which packageretrovirus. Viral vectors used in gene therapy are usually generated byproducing a cell line that packages a nucleic acid vector into a viralparticle. The vectors typically contain the minimal viral sequencesrequired for packaging and subsequent integration into a host, otherviral sequences being replaced by an expression cassette for thepolynucleotide(s) to be expressed. The missing viral functions aretypically supplied in trans by the packaging cell line. For example, AAVvectors used in gene therapy typically only possess ITR sequences fromthe AAV genome which are required for packaging and integration into thehost genome. Viral DNA is packaged in a cell line, which contains ahelper plasmid encoding the other AAV genes, namely rep and cap, butlacking ITR sequences. The cell line may also be infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV. Additionalmethods for the delivery of nucleic acids to cells are known to thoseskilled in the art. See, for example, US20030087817, incorporated hereinby reference.

In some embodiments, a host cell is transiently or non-transientlytransfected with one or more vectors described herein. In someembodiments, a cell is transfected as it naturally occurs in a subject.In some embodiments, a cell that is transfected is taken from a subject.In some embodiments, the cell is derived from cells taken from asubject, such as a cell line. A wide variety of cell lines for tissueculture are known in the art. Examples of cell lines include, but arenot limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1,Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1,CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480,SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E,MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss,3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T,3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549,ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3,C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T,CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7,COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3,EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa,Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812,KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231,MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A,MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3,NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F,RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line,U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, andtransgenic varieties thereof. Cell lines are available from a variety ofsources known to those with skill in the art (see, e.g., the AmericanType Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, acell transfected with one or more vectors described herein is used toestablish a new cell line comprising one or more vector-derivedsequences. In some embodiments, a cell transiently transfected with thecomponents of a nucleic acid-targeting system as described herein (suchas by transient transfection of one or more vectors, or transfectionwith RNA), and modified through the activity of a nucleic acid-targetingcomplex, is used to establish a new cell line comprising cellscontaining the modification but lacking any other exogenous sequence. Insome embodiments, cells transiently or non-transiently transfected withone or more vectors described herein, or cell lines derived from suchcells are used in assessing one or more test compounds.

In some embodiments, one or more vectors described herein are used toproduce a non-human transgenic animal or transgenic plant. In someembodiments, the transgenic animal is a mammal, such as a mouse, rat, orrabbit. In certain embodiments, the organism or subject is a plant. Incertain embodiments, the organism or subject or plant is algae. Methodsfor producing transgenic plants and animals are known in the art, andgenerally begin with a method of cell transfection, such as describedherein.

In one aspect, the invention provides for methods of modifying a targetpolynucleotide in a eukaryotic cell. In some embodiments, the methodcomprises allowing a nucleic acid-targeting complex to bind to thetarget polynucleotide to effect cleavage of said target polynucleotidethereby modifying the target polynucleotide, wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said target polynucleotide.

In one aspect, the invention provides a method of modifying expressionof a polynucleotide in a eukaryotic cell. In some embodiments, themethod comprises allowing a nucleic acid-targeting complex to bind tothe polynucleotide such that said binding results in increased ordecreased expression of said polynucleotide; wherein the nucleicacid-targeting complex comprises a nucleic acid-targeting effectorprotein complexed with a guide RNA hybridized to a target sequencewithin said polynucleotide.

C2c2 Effector Protein Complexes can be Used in Plants

The C2c2 effector protein system(s) (e.g., single or multiplexed) can beused in conjunction with recent advances in crop genomics. The systemsdescribed herein can be used to perform efficient and cost effectiveplant gene or genome interrogation or editing or manipulation—forinstance, for rapid investigation and/or selection and/or interrogationsand/or comparison and/or manipulations and/or transformation of plantgenes or genomes; e.g., to create, identify, develop, optimize, orconfer trait(s) or characteristic(s) to plant(s) or to transform a plantgenome. There can accordingly be improved production of plants, newplants with new combinations of traits or characteristics or new plantswith enhanced traits. The C2c2 effector protein system(s) can be usedwith regard to plants in Site-Directed Integration (SDI) or Gene Editing(GE) or any Near Reverse Breeding (NRB) or Reverse Breeding (RB)techniques. Aspects of utilizing the herein described C2c2 effectorprotein systems may be analogous to the use of the CRISPR-Cas (e.g.CRISPR-Cas9) system in plants, and mention is made of the University ofArizona website “CRISPR-PLANT” (http://www.genome.arizona.edu/crispr/)(supported by Penn State and AGI). Embodiments of the invention can beused in genome editing in plants or where RNAi or similar genome editingtechniques have been used previously; see, e.g., Nekrasov, “Plant genomeediting made easy: targeted mutagenesis in model and crop plants usingthe CRISPR-Cas system,” Plant Methods 2013, 9:39(doi:10.1186/1746-4811-9-39); Brooks, “Efficient gene editing in tomatoin the first generation using the CRISPR-Cas9 system,” Plant PhysiologySeptember 2014 pp 114.247577; Shan, “Targeted genome modification ofcrop plants using a CRISPR-Cas system,” Nature Biotechnology 31, 686-688(2013); Feng, “Efficient genome editing in plants using a CRISPR/Cassystem,” Cell Research (2013) 23:1229-1232. doi:10.1038/cr.2013.114;published online 20 Aug. 2013; Xie, “RNA-guided genome editing in plantsusing a CRISPR-Cas system,” Mol Plant. 2013 November; 6(6):1975-83. doi:10.1093/mp/sst119. Epub 2013 Aug. 17; Xu, “Gene targeting using theAgrobacterium tumefaciens-mediated CRISPR-Cas system in rice,” Rice2014, 7:5 (2014), Zhou et al., “Exploiting SNPs for biallelic CRISPRmutations in the outcrossing woody perennial Populus reveals4-coumarate: CoA ligase specificity and Redundancy,” New Phytologist(2015) (Forum) 1-4 (available online only at www.newphytologist.com);Caliando et al., “Targeted DNA degradation using a CRISPR device stablycarried in the host genome, NATURE COMMUNICATIONS 6:6989, DOI:10.1038/ncomms7989, www.nature.com/naturecommunications DOI:10.1038/ncomms7989; U.S. Pat. No. 6,603,061 —Agrobacterium-MediatedPlant Transformation Method; U.S. Pat. No. 7,868,149—Plant GenomeSequences and Uses Thereof and US 2009/0100536—Transgenic Plants withEnhanced Agronomic Traits, all the contents and disclosure of each ofwhich are herein incorporated by reference in their entirety. In thepractice of the invention, the contents and disclosure of Morrell et al.“Crop genomics: advances and applications,” Nat Rev Genet. 2011 Dec. 29;13(2):85-96; each of which is incorporated by reference herein includingas to how herein embodiments may be used as to plants. Accordingly,reference herein to animal cells may also apply, mutatis mutandis, toplant cells unless otherwise apparent; and, the enzymes herein havingreduced off-target effects and systems employing such enzymes can beused in plant applications, including those mentioned herein.

Sugano et al. (Plant Cell Physiol. 2014 March; 55(3):475-81. doi:10.1093/pcp/pcu014. Epub 2014 Jan. 18) reports the application ofCRISPR-Cas9 to targeted mutagenesis in the liverwort Marchantiapolymorpha L., which has emerged as a model species for studying landplant evolution. The U6 promoter of M. polymorpha was identified andcloned to express the gRNA. The target sequence of the gRNA was designedto disrupt the gene encoding auxin response factor 1 (ARF1) in M.polymorpha. Using Agrobacterium-mediated transformation, Sugano et al.isolated stable mutants in the gametophyte generation of M. polymorpha.CRISPR/Cas9-based site-directed mutagenesis in vivo was achieved usingeither the Cauliflower mosaic virus 35S or M. polymorpha EF1α promoterto express Cas9. Isolated mutant individuals showing an auxin-resistantphenotype were not chimeric. Moreover, stable mutants were produced byasexual reproduction of T1 plants. Multiple arf1 alleles were easilyestablished using CRIPSR/Cas9-based targeted mutagenesis. The C2c2systems of the present invention can be used to regulate the same aswell as other genes, and like expression control systems such as RNAiand siRNA, the method of the invention can be inducible and reversible.

Kabadi et al. (Nucleic Acids Res. 2014 Oct. 29; 42(19):e147. doi:10.1093/nar/gku749. Epub 2014 Aug. 13) developed a single lentiviralsystem to express a Cas9 variant, a reporter gene and up to four sgRNAsfrom independent RNA polymerase III promoters that are incorporated intothe vector by a convenient Golden Gate cloning method. Each sgRNA wasefficiently expressed and can mediate multiplex gene editing andsustained transcriptional activation in immortalized and primary humancells. The instant invention can be used to regulate the plant genes ofKabadi.

Xing et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9binary vector set based on the pGreen or pCAMBIA backbone, as well as agRNA. This toolkit requires no restriction enzymes besides BsaI togenerate final constructs harboring maize-codon optimized Cas9 and oneor more gRNAs with high efficiency in as little as one cloning step. Thetoolkit was validated using maize protoplasts, transgenic maize lines,and transgenic Arabidopsis lines and was shown to exhibit highefficiency and specificity. More importantly, using this toolkit,targeted mutations of three Arabidopsis genes were detected intransgenic seedlings of the T1 generation. Moreover, the multiple-genemutations could be inherited by the next generation. (guide RNA)modulevector set, as a toolkit for multiplex genome editing in plants. TheC2c2 systems and proteins of the instant invention may be used to targetthe genes targeted by Xing.

The C2c2 CRISPR systems of the invention may be used in the detection ofplant viruses. Gambino et al. (Phytopathology. 2006 November;96(11):1223-9. doi: 10.1094/PHYTO-96-1223) relied on amplification andmultiplex PCR for simultaneous detection of nine grapevine viruses. TheC2c2 systems and proteins of the instant invention may similarly be usedto detect multiple targets in a host. Moreover, the systems of theinvention can be used to simultaneously knock down viral gene expressionin valuable cultivars, and prevent activation or further infection bytargeting expressed viral RNA.

Murray et al. (Proc Biol Sci. 2013 Jun. 26; 280(1765):20130965. doi:10.1098/rspb.2013.0965; published 2013 Aug. 22) analyzed 12 plant RNAviruses to investigate evolutionary rates and found evidence of episodicselection possibly due to shifts between different host genotypes orspecies. The C2c2 systems and proteins of the instant invention may beused to target or immunize against such viruses in a host. For example,the systems of the invention can be used to block viral RNA expressionhence replication. Also, the invention can be used to target nucleicacids for cleavage as well as to target expression or activation.Moreover, the systems of the invention can be multiplexed so as to hitmultiple targets or multiple isolate of the same virus.

Ma et al. (Mol Plant. 2015 Aug. 3; 8(8):1274-84. doi:10.1016/j.molp.2015.04.007) reports robust CRISPR-Cas9 vector system,utilizing a plant codon optimized Cas9 gene, for convenient andhigh-efficiency multiplex genome editing in monocot and dicot plants. Maet al. designed PCR-based procedures to rapidly generate multiple sgRNAexpression cassettes, which can be assembled into the binary CRISPR-Cas9vectors in one round of cloning by Golden Gate ligation or GibsonAssembly. With this system, Ma et al. edited 46 target sites in ricewith an average 85.4% rate of mutation, mostly in biallelic andhomozygous status. Ma et al. provide examples of loss-of-function genemutations in TO rice and T1 Arabidopsis plants by simultaneous targetingof multiple (up to eight) members of a gene family, multiple genes in abiosynthetic pathway, or multiple sites in a single gene. Similarly, theC2c2 systems of the instant invention can dffieicnelty target expressionof multiple genes simultaneously.

Lowder et al. (Plant Physiol. 2015 Aug. 21. pii: pp. 00636.2015) alsodeveloped a CRISPR-Cas9 toolbox enables multiplex genome editing andtranscriptional regulation of expressed, silenced or non-coding genes inplants. This toolbox provides researchers with a protocol and reagentsto quickly and efficiently assemble functional CRISPR-Cas9 T-DNAconstructs for monocots and dicots using Golden Gate and Gateway cloningmethods. It comes with a full suite of capabilities, includingmultiplexed gene editing and transcriptional activation or repression ofplant endogenous genes. T-DNA based transformation technology isfundamental to modern plant biotechnology, genetics, molecular biologyand physiology. As such, we developed a method for the assembly of Cas9(WT, nickase or dCas9) and gRNA(s) into a T-DNA destination-vector ofinterest. The assembly method is based on both Golden Gate assembly andMultiSite Gateway recombination. Three modules are required forassembly. The first module is a Cas9 entry vector, which containspromoterless Cas9 or its derivative genes flanked by attL1 and attR5sites. The second module is a gRNA entry vector which contains entrygRNA expression cassettes flanked by attL5 and attL2 sites. The thirdmodule includes attR1-attR2-containing destination T-DNA vectors thatprovide promoters of choice for Cas9 expression. The toolbox of Lowderet al. may be applied to the C2c2 effector protein system of the presentinvention.

Organisms such as yeast and microalgae are widely used for syntheticbiology. Stovicek et al. (Metab. Eng. Comm., 2015; 2:13) describesgenome editing of industrial yeast, for example, Saccharomycescerevisiae, to efficiently produce robust strains for industrialproduction. Stovicek used a CRISPR-Cas9 system codon-optimized for yeastto simultaneously disrupt both alleles of an endogenous gene and knockin a heterologous gene. Cas9 and gRNA were expressed from genomic orepisomal 2μ based vector locations. The authors also showed that genedisruption efficiency could be improved by optimization of the levels ofCas9 and gRNA expression. Hlavová et al. (Biotechnol. Adv. 2015)discusses development of species or strains of microalgae usingtechniques such as CRISPR to target nuclear and chloroplast genes forinsertional mutagenesis and screening. The same plasmids and vectors canbe applied to the C2c2 systems of the instant invention.

Petersen (“Towards precisely glycol engineered plants,” Plant BiotechDenmark Annual meeting 2015, Copenhagen, Denmark) developed a method ofusing CRISPR/Cas9 to engineer genome changes in Arabidopsis, for exampleto applied Arabidopsis for production of proteins and products havingdesired posttranslational modifications. Hebelstrup et al. (Front PlantSci. 2015 Apr. 23; 6:247) outlines in planta starch bioengineering,providing crops that express starch modifying enzymes and directlyproduce products that normally are made by industrial chemical and/orphysical treatments of starches. The methods of Petersen and Hebelstrupmay be applied to the C2c2 effector protein system of the presentinvention.

Kurth et al., (J Virol. 2012 June; 86(11):6002-9. Doi:10.1128/JVI.00436-12. Epub 2012 Mar. 21) developed an RNA virus-basedvector for the introduction of desired traits into grapevine withoutheritable modifications to the genome. The vector provided the abilityto regulate expression of endogenous genes by virus-induced genesilencing. The C2c2 systems and proteins of the instant invention can beused to silence genes and proteins without heritable modification to thegenome.

In an embodiment, the plant may be a legume. The present invention mayutilize the herein disclosed CRISP-Cas system for exploring andmodifying, for example, without limitation, soybeans, peas, and peanuts.Curtin et al. provides a toolbox for legume function genomics. (SeeCurtin et al., “A genome engineering toolbox for legume Functionalgenomics,” International Plant and Animal Genome Conference XXII 2014).Curtin used the genetic transformation of CRISPR to knock-out/downsingle copy and duplicated legume genes both in hairy root and wholeplant systems. Some of the target genes were chosen in order to exploreand optimize the features of knock-out/down systems (e.g., phytoenedesaturase), while others were identified by soybean homology toArabidopsis Dicer-like genes or by genome-wide association studies ofnodulation in Medicago. The C2c2 systems and proteins of the instantinvention can be used to knockout/knockdown systems.

Peanut allergies and allergies to legumes generally are a real andserious health concern. The C2c2 effector protein system of the presentinvention can be used to identify and then edit or silence genesencoding allergenic proteins of such legumes. Without limitation as tosuch genes and proteins, Nicolaou et al. identifies allergenic proteinsin peanuts, soybeans, lentils, peas, lupin, green beans, and mung beans.(See, Nicolaou et al., Current Opinion in Allergy and ClinicalImmunology 2011; 11(3):222).

In an advantageous embodiment, the plant may be a tree. The presentinvention may also utilize the herein disclosed CRISPR Cas system forherbaceous systems (see, e.g., Belhaj et al., Plant Methods 9: 39 andHarrison et al., Genes & Development 28: 1859-1872). In a particularlyadvantageous embodiment, the CRISPR Cas system of the present inventionmay target single nucleotide polymorphisms (SNPs) in trees (see, e.g.,Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301,October 2015). In the Zhou et al. study, the authors applied a CRISPRCas system in the woody perennial Populus using the 4-coumarate:CoAligase (4CL) gene family as a case study and achieved 100% mutationalefficiency for two 4CL genes targeted, with every transformant examinedcarrying biallelic modifications. In the Zhou et al., study, theCRISPR-Cas9 system was highly sensitive to single nucleotidepolymorphisms (SNPs), as cleavage for a third 4CL gene was abolished dueto SNPs in the target sequence. These methods may be applied to the C2c2effector protein system of the present invention.

The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages298-301, October 2015) may be applied to the present invention asfollows. Two 4CL genes, 4CL1 and 4CL2, associated with lignin andflavonoid biosynthesis, respectively are targeted for CRISPR-Cas9editing. The Populus tremula×alba clone 717-1B4 routinely used fortransformation is divergent from the genome-sequenced Populustrichocarpa. Therefore, the 4CL1 and 4CL2 gRNAs designed from thereference genome are interrogated with in-house 717 RNA-Seq data toensure the absence of SNPs which could limit Cas efficiency. A thirdgRNA designed for 4CL5, a genome duplicate of 4CL1, is also included.The corresponding 717 sequence harbors one SNP in each allelenear/within the PAM, both of which are expected to abolish targeting bythe 4CL5-gRNA. All three gRNA target sites are located within the firstexon. For 717 transformation, the gRNA is expressed from the MedicagoU6.6 promoter, along with a human codon-optimized Cas under control ofthe CaMV 35S promoter in a binary vector. Transformation with theCas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2lines are subjected to amplicon-sequencing. The data is then processedand biallelic mutations are confirmed in all cases. These methods may beapplied to the C2c2 effector protein system of the present invention.

In plants, pathogens are often host-specific. For example, Fusariumoxysporum f. sp. Lycopersici causes tomato wilt but attacks only tomato,and F. oxysporum f. dianthi Puccinia graminis f. sp. Tritici attacksonly wheat. Plants have existing and induced defenses to resist mostpathogens. Mutations and recombination events across plant generationslead to genetic variability that gives rise to susceptibility,especially as pathogens reproduce with more frequency than plants. Inplants there can be non-host resistance, e.g., the host and pathogen areincompatible. There can also be Horizontal Resistance, e.g., partialresistance against all races of a pathogen, typically controlled by manygenes and Vertical Resistance, e.g., complete resistance to some racesof a pathogen but not to other races, typically controlled by a fewgenes. In a Gene-for-Gene level, plants and pathogens evolve together,and the genetic changes in one balance changes in other. Accordingly,using Natural Variability, breeders combine most useful genes for Yield,Quality, Uniformity, Hardiness, Resistance. The sources of resistancegenes include native or foreign Varieties, Heirloom Varieties, WildPlant Relatives, and Induced Mutations, e.g., treating plant materialwith mutagenic agents. Using the present invention, plant breeders areprovided with a new tool to induce mutations. Accordingly, one skilledin the art can analyze the genome of sources of resistance genes, and inVarieties having desired characteristics or traits employ the presentinvention to induce the rise of resistance genes, with more precisionthan previous mutagenic agents and hence accelerate and improve plantbreeding programs.

Aside from the plants otherwise discussed herein and above, engineeredplants modified by the effector protein and suitable guide, and progenythereof, as provided. These may include disease or drought resistantcrops, such as wheat, barley, rice, soybean or corn; plants modified toremove or reduce the ability to self-pollinate (but which can instead,optionally, hybridise instead); and allergenic foods such as peanuts andnuts where the immunogenic proteins have been disabled, destroyed ordisrupted by targeting via a effector protein and suitable guide.

Therapeutic Treatment

The system of the invention can be applied in areas of former RNAcutting technologies, without undue experimentation, from thisdisclosure, including therapeutic, assay and other applications, becausethe present application provides the foundation for informed engineeringof the system. The present invention provides for therapeutic treatmentof a disease caused by overexpression of RNA, toxic RNA and/or mutatedRNA (such as, for example, splicing defects or truncations). Expressionof the toxic RNA may be associated with formation of nuclear inclusionsand late-onset degenerative changes in brain, heart or skeletal muscle.In the best studied example, myotonic dystrophy, It appears that themain pathogenic effect of the toxic RNA is to sequester binding proteinsand compromise the regulation of alternative splicing (Hum. Mol. Genet.(2006) 15 (suppl 2): R162-R169). Myotonic dystrophy [dystrophiamyotonica (DM)] is of particular interest to geneticists because itproduces an extremely wide range of clinical features. A partial listingwould include muscle wasting, cataracts, insulin resistance, testicularatrophy, slowing of cardiac conduction, cutaneous tumors and effects oncognition. The classical form of DM, which is now called DM type 1(DM1), is caused by an expansion of CTG repeats in the 3′-untranslatedregion (UTR) of DMPK, a gene encoding a cytosolic protein kinase.

The below table presents a list of exons shown to have misregulatedalternative splicing in DM1 skeletal muscle, heart or brain.

Tissue/gene Target Skeletal muscle Reference ALP ex 5a, 5b Lin X., etal. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 CAPN3 ex 16 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 CLCN1 int 2, ex7a, 8a Mankodi A., et al. Expanded CUG repeats trigger aberrant splicingof ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletalmuscle in myotonic dystrophy. Mol. Cell 2002; 10: 35-44 Charlet-B N., etal. Loss of the muscle-specific chloride channel in type 1 myotonicdystrophy due to misregulated alternative splicing. Mol. Cell 2002; 10:45-53 FHOS ex 11a Lin X., et al. Failure of MBNL1-dependent postnatalsplicing transitions in myotonic dystrophy. Hum. Mol. Genet 2006; 15:2087-2097 GFAT1 ex 10 Lin X., et al. Failure of MBNL1-dependentpostnatal splicing transitions in myotonic dystrophy. Hum. Mol. Genet2006; 15: 2087-2097 IR ex 11 Savkur R. S., et al. Aberrant regulation ofinsulin receptor alternative splicing is associated with insulinresistance in myotonic dystrophy. Nat. Genet. 2001; 29: 40-47 MBNL1 ex 7Lin X., et al. Failure of MBNL1-dependent postnatal splicing transitionsin myotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 MBNL2 ex 7Lin X., et al. Failure of MBNL1-dependent postnatal splicing transitionsin myotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 MTMR1 ex 2.1,2.2 Buj-Bello A., et al. Muscle-specific alternative splicing ofmyotubularin-related 1 gene is impaired in DM1 muscle cells. Hum. Mol.Genet. 2002; 11: 2297-2307 NRAP ex 12 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006; 15: 2087-2097 RYR1 ex 70 Kimura T., et al. AlteredmRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005; 14: 2189-2200 SERCA1 ex 22 Kimura T., etal. Altered mRNA splicing of the skeletal muscle ryanodine receptor andsarcoplasmic/endoplasmic reticulum Ca2+- ATPase in myotonic dystrophytype 1. Hum. Mol. Genet. 2005; 14: 2189-2200 Lin X., et al. Failure ofMBNL1-dependent postnatal splicing transitions in myotonic dystrophy.Hum. Mol. Genet 2006; 15: 2087-2097 z-Titin ex Zr4, Zr5 Lin X., et al.Failure of MBNL1-dependent postnatal splicing transitions in myotonicdystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 m-Titin M-line ex5 LinX., et al. Failure of MBNL1-dependent postnatal splicing transitions inmyotonic dystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 TNNT3 fetal exKanadia R. N., et al. A muscleblind knockout model for myotonicdystrophy. Science 2003; 302: 1978-1980 ZASP ex 11 Lin X., et al.Failure of MBNL1-dependent postnatal splicing transitions in myotonicdystrophy. Hum. Mol. Genet 2006; 15: 2087-2097 Heart TNNT2 ex 5 PhilipsA. V., et al. Disruption of splicing regulated by a CUG- binding proteinin myotonic dystrophy. Science 1998; 280: 737-741 ZASP ex 11 Mankodi A.,et al. Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res.2005; 97: 1152-1155 m-Titin M-line ex 5 Mankodi A., et al. Nuclear RNAfoci in the heart in myotonic dystrophy. Circ. Res. 2005; 97: 1152-1155KCNAB1 ex 2 Mankodi A., et al. Nuclear RNA foci in the heart in myotonicdystrophy. Circ. Res. 2005; 97: 1152-1155 ALP ex 5 (Mankodi A., et al.Nuclear RNA foci in the heart in myotonic dystrophy. Circ. Res. 2005;97: 1152-1155 Brain TAU ex 2, ex 10 Sergeant N., et al. Dysregulation ofhuman brain microtubule- associated tau mRNA maturation in myotonicdystrophy type 1. Hum. Mol. Genet. 2001; 10: 2143-2155 Jiang H., et al.Myotonic dystrophy type 1 associated with nuclear foci of mutant RNA,sequestration of muscleblind proteins, and deregulated alternativesplicing in neurons. Hum. Mol. Genet. 2004; 13: 3079-3088 APP ex 7 JiangH., et al. Myotonic dystrophy type 1 associated with nuclear foci ofmutant RNA, sequestration of muscleblind proteins, and deregulatedalternative splicing in neurons. Hum. Mol. Genet. 2004; 13: 3079-3088NMDAR1 ex 5 Jiang H., et al. Myotonic dystrophy type 1 associated withnuclear foci of mutant RNA, sequestration of muscleblind proteins, andderegulated alternative splicing in neurons. Hum. Mol. Genet. 2004; 13:3079-3088

The enzymes of the present invention may target overexpressed RNA ortoxic RNA, such as for example, the DMPK gene or any of the misregulatedalternative splicing in DM1 skeletal muscle, heart or brain in, forexample, the above table.

The enzymes of the present invention may also target trans-actingmutations affecting RNA-dependent functions that cause disease(summarized in Cell. 2009 Feb. 20; 136(4): 777-793) as indicated in thebelow table.

DISEASE GENE/MUTATION FUNCTION Prader Willi syndrome SNORD116 ribosomebiogenesis Spinal muscular atrophy (SMA) SMN2 splicing Dyskeratosiscongenita (X-linked) DKC1 telomerase/translation Dyskeratosis congenita(autosomal TERC telomerase dominant) Dyskeratosis congenita (autosomalTERT telomerase dominant) Diamond-Blackfan anemia RPS19, RPS24 ribosomebiogenesis Shwachman-Diamond syndrome SBDS ribosome biogenesisTreacher-Collins syndrome TCOF1 ribosome biogenesis Prostate cancerSNHG5 ribosome biogenesis Myotonic dystrophy, type 1 (DM1) DMPK (RNAgain-of- protein kinase function) Myotonic dystrophy type 2 (DM2) ZNF9(RNA gain-of- RNA binding function) Spinocerebellar ataxia 8 (SCA8)ATXN8/ATXN8OS (RNA unknown/noncoding gain-of-function) RNA Huntington'sdisease-like 2 (HDL2) JPH3 (RNA gain-of- ion channel function function)Fragile X-associated tremor ataxia FMR1 (RNA gain-of- translation/mRNAsyndrome (FXTAS) function) localization Fragile X syndrome FMR1translation/mRNA localization X-linked mental retardation UPF3Btranslation/nonsense mediated decay Oculopharyngeal muscular dystrophyPABPN1 3′ end formation (OPMD) Human pigmentary genodermatosis DSRADediting Retinitis pigmentosa PRPF31 splicing Retinitis pigmentosa PRPF8splicing Retinitis pigmentosa HPRP3 splicing Retinitis pigmentosa PAP1splicing Cartilage-hair hypoplasia (recessive) RMRP splicing Autism7q22-q33 locus breakpoint noncoding RNA Beckwith-Wiedemann syndrome H19noncoding RNA (BWS) Charcot-Marie-Tooth (CMT) Disease GRS translationCharcot-Marie-Tooth (CMT) Disease YRS translation Amyotrophic lateralsclerosis (ALS) TARDBP splicing, transcription Leukoencephalopathy withvanishing EIF2B1 translation white matter Wolcott-Rallison syndromeEIF2AK3 translation (protease) Mitochondrial myopathy and PUS1translation sideroblastic anemia (MLASA) Encephalomyopathy andhypertrophic TSFM translation cardiomyopathy (mitochondrial) Hereditaryspastic paraplegia SPG7 ribosome biogenesis Leukoencephalopathy DARS2translation (mitochondrial) Susceptibility to diabetes mellitus LARS2translation (mitochondrial) Deafness MTRNR1 ribosome biogenesis(mitochondrial) MELAS syndrome, deafness MTRNR2 ribosome biogenesis(mitochondrial) Cancer SFRS1 splicing, translation, export Cancer RBM5splicing Multiple disorders mitochondrial tRNA translation mutations(mitochondrial) Cancer miR-17-92 cluster RNA interference CancermiR-372/miR-373 RNA interference

The enzyme of the present invention may also be used in the treatment ofvarious tauopathies, including primary and secondary tauopathies, suchas primary age-related tauopathy (PART)/Neurofibrillarytangle-predominant senile dementia, with NFTs similar to AD, but withoutplaques, dementia pugilistica (chronic traumatic encephalopathy),progressive supranuclear palsy, corticobasal degeneration,frontotemporal dementia and parkinsonism linked to chromosome 17,lytico-Bodig disease (Parkinson-dementia complex of Guam), gangliogliomaand gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism,subacute sclerosing panencephalitis, as well as lead encephalopathy,tuberous sclerosis, Hallervorden-Spatz disease, and lipofuscinosis,Alzheime's disease. The enzymes of the present invention may also targetmutations disrupting the cis-acting splicing code cause splicing defectsand disease (summarized in Cell. 2009 Feb. 20; 136(4): 777-793). Themotor neuron degenerative disease SMA results from deletion of the SMN1gene. The remaining SMN2 gene has a C→T substitution in exon 7 thatinactivates an exonic splicing enhancer (ESE), and creates an exonicsplicing silencer (ESS), leading to exon 7 skipping and a truncatedprotein (SMNA7). A T→A substitution in exon 31 of the dystrophin genesimultaneously creates a premature termination codon (STOP) and an ESS,leading to exon 31 skipping. This mutation causes a mild form of DMDbecause the mRNA lacking exon 31 produces a partially functionalprotein. Mutations within and downstream of exon 10 of the MAPT geneencoding the tau protein affect splicing regulatory elements and disruptthe normal 1:1 ratio of mRNAs including or excluding exon 10. Thisresults in a perturbed balance between tau proteins containing eitherfour or three microtubule-binding domains (4R-tau and 3R-tau,respectively), causing the neuropathological disorder FTDP-17. Theexample shown is the N279K mutation which enhances an ESE functionpromoting exon 10 inclusion and shifting the balance toward increased4R-tau. Polymorphic (UG)m(U)n tracts within the 3′ splice site of theCFTR gene exon 9 influence the extent of exon 9 inclusion and the levelof full-length functional protein, modifying the severity of cysticfibrosis (CF) caused by a mutation elsewhere in the CFTR gene.

The innate immune system detects viral infection primarily byrecognizing viral nucleic acids inside an infected cell, referred to asDNA or RNA sensing. In vitro RNA sensing assays can be used to detectspecific RNA substrates. The RNA targeting effector protein can forinstance be used for RNA-based sensing in living cells. Examples ofapplications are diagnostics by sensing of, for examples,disease-specific RNAs.

The RNA targeting effector protein of the invention can further be usedfor antiviral activity, in particular against RNA viruses. The effectorprotein can be targeted to the viral RNA using a suitable guide RNAselective for a selected viral RNA sequence. In particular, the effectorprotein may be an active nuclease that cleaves RNA, such as singlestranded RNA. Provided is therefore the use of an RNA targeting effectorprotein of the invention as an antiviral agent.

Therapeutic dosages of the enzyme system of the present invention totarget RNA the above-referenced RNAs are contemplated to be about 0.1 toabout 2 mg/kg the dosages may be administered sequentially with amonitored response, and repeated dosages if necessary, up to about 7 to10 doses per patient. Advantageously, samples are collected from eachpatient during the treatment regimen to ascertain the effectiveness oftreatment. For example, RNA samples may be isolated and quantified todetermine if expression is reduced or ameliorated. Such a diagnostic iswithin the purview of one of skill in the art.

Transcript Detection Methods

The effector proteins and systems of the invention are useful forspecific detection of RNAs in a cell or other sample. In the presence ofan RNA target of interest, guide-dependent C2c2 nuclease activity may beaccompanied by non-specific RNAse activity against collateral targets.To take advantage of the rNase activity, all that is needed is areporter substrate that can be detectably cleaved. For example, areporter molecule can comprise RNA, tagged with a fluorescent reportermolecule (fluor) on one end and a quencher on the other. In the absenceof C2c2 rNase activity, the physical proximity of the quencher dampensfluorescence from the fluor to low levels. When C2c2 target specificcleavage is activated by the presence of an RNA target-of-interest andsuitable guide RNA, the RNA-containing reporter molecule isnon-specifically cleaved and the fluor and quencher are spatiallyseparated. This causes the fluor to emit a detectable signal whenexcited by light of the appropriate wavelength.

In an aspect, the invention relates to a (target) RNA detection systemcomprising an RNA targeting effector; one or more guide RNAs designed tobind to the corresponding RNA target; and an RNA-based cleavageinducible reporter construct. In another aspect, the invention relatesto a method for (target) RNA detection in a sample, comprising adding anRNA targeting effector, one or more guide RNAs designed to bind to said(target) RNA, and an RNA-based cleavage inducible reporter construct tosaid sample. In a further aspect, the invention relates to a kit ordevice comprising the (target) RNA detection system as defined herein,or a kit or device comprising at least the RNA targeting effector andthe RNA-based cleavage inducible reporter construct. In a furtheraspect, the invention relates to the use of the RNA targeting system orkit or device as defined herein for (target) RNA detection. The RNAtargeting effector in certain embodiments is an RNA guided RNAse. Incertain embodiments, the RNA targeting effector is a CRISPR effector. Incertain embodiments, the RNA targeting effector is a class 2 CRISPReffector. In certain embodiments, the RNA targeting effector is a class2, type VI CRISPR effector. In a preferred embodiment, the RNA targetingeffector is C2c2. In certain embodiments, the RNA targeting effector,preferably C2c2, is derived from a species as described hereinelsewhere. It will be understood that the guide RNA designed to bind tosaid (target) RNA as described herein is capable of forming a complexwith the RNA targeting effector and wherein the guide RNA in saidcomplex is capable of binding to a target RNA molecule and whereby thetarget RNA is cleaved, as also described herein elsewhere. It will beunderstood that the guide RNA typically comprises a guide sequence and adirect repeat, as described herein elsewhere. In certain embodiments,the one or more guide RNAs are designed to bind to one or more targetmolecules that are diagnostic for a disease state. In certainembodiments, the disease state is infection, such as viral, bacterial,fungal, or parasitic infection. In certain embodiments, the diseasestate is characterised by aberrant (target) RNA expression. In certainembodiments, the disease state is cancer. In certain embodiments, thedisease state is autoimmune disease. The RNA-based cleavage induciblereporter construct comprises RNA and cleavage of the RNA results in adetectable readout, i.e. a detectable signal is generated upon cleavageof the RNA. In certain embodiments, the RNA-based cleavage induciblereporter construct comprises a fluorochrome and a quencher. The skilledperson will understand that different types of fluorochromes andcorresponding quenchers may be used. The skilled person will readilyenvisage other types of inducible reporter systems which may be adaptedfor use in the present RNA cleavage reporter constructs.

In one exemplary assay method, C2c2 effector,target-of-interest-specific guide RNA, and reporter molecule are addedto a cellular sample. An increase in fluorescence indicates the presenceof the RNA target-of-interest. In another exemplary method, a detectionarray is provided. Each location of the array is provided with C2c2effector, reporter molecule, and a target-of-interest-specific guideRNA. Depending on the assay to be performed, thetarget-of-interest-specific guide RNAs at each location of the array canbe the same, different, or a combination thereof. Differenttarget-of-interest-specific guide RNAs might be provided, for examplewhen it is desired to test for one or more targets in a single sourcesample. The same target-of-interest-specific guide RNA might be providedat each location, for example when it is desired to test multiplesamples for the same target.

As used herein, a “masking construct” refers to a molecule that can becleaved or otherwise deactivated by an activated CRISPR system effectorprotein described herein. In certain example embodiments, the maskingconstruct is a RNA-based masking construct. The masking constructprevents the generation or detection of a positive detectable signal. Apositive detectable signal may be any signal that can be detected usingoptical, fluorescent, chemiluminescent, electrochemical or otherdetection methods known in the art. The masking construct may preventthe generation of a detectable positive signal or mask the presence of adetectable positive signal until the masking construct is removed orotherwise silenced. The term “positive detectable signal” is used todifferentiate from other detectable signals that may be detectable inthe presence of the masking construct. For example, in certainembodiments a first signal may be detected when the masking agent ispresent (i.e. a negative detectable signal), which then converts to asecond signal (e.g. the positive detectable signal) upon detection ofthe target molecules and cleavage or deactivation of the masking agentby the activated CRISPR effector protein.

In certain example embodiments, the masking construct may suppressgeneration of a gene product. The gene product may be encoded by areporter construct that is added to the sample. The masking constructmay be an interfering RNA involved in a RNA interference pathway, suchas a shRNA or siRNA. The masking construct may also comprise microRNA(miRNA). While present, the masking construct suppresses expression ofthe gene product. The gene product may be a fluorescent protein or otherRNA transcript or proteins that would otherwise be detectable by alabeled probe or antibody but for the presence of the masking construct.Upon activation of the effector protein the masking construct is cleavedor otherwise silenced allowing for expression and detection of the geneproduct as the positive detectable signal.

In certain example embodiments, the masking construct may sequester oneor more reagents needed to generate a detectable positive signal suchthat release of the one or more reagents from the masking constructresults in generation of the detectable positive signal. The one or morereagents may combine to produce a colorimetric signal, achemiluminescent signal, a fluorescent signal, or any other detectablesignal and may comprise any reagents known to be suitable for such apurpose. In certain example embodiments, the one or more reagents aresequestered by RNA aptamers that bind the one or more reagents. The oneor more reagents are released when the effector protein is activatedupon detection of a target molecule. In certain example embodiments, theone or more reagents is a protein, such as an enzyme, capable offacilitating generation of a detectable signal, such as a colorimetric,chemiluminescent, or fluorescent signal, that is inhibited orsequestered such that the protein cannot generate the detectable signalby the binding of one or more RNA aptamers to the protein. Uponactivation of the effector proteins disclosed herein, the RNA aptamersare cleaved or degraded to the extent they no longer inhibit theprotein's ability to generate the detectable signal.

In one embodiment, thrombin is used as a signal amplification enzymewith an inhibitory aptamer, for example having the following sequence:GGGAACAAAGCUGAAGUACUUACCC (SEQ ID NO: 56). When this aptamer is cleaved,thrombin becomes active and will cleave a peptide colorimetric substrate(see, e.g.,www.sigmaaldrich.com/catalog/product/sigma/t3068?lang=en&region=US) orfluorescent substrate (see, e.g.,www.sigmaaldrich.com/catalog/product/sigma/b9385?lang=en&region=US). Thecolorimetric substrate, para-nitroaniline (pNA), is covalently linked tothe peptide substrate for thrombin. Upon cleavage by thrombin, pNA isreleased and becomes yellow in color and easily visible by eye. Thefluorescent substrate operates by a similar principle and, upon cleavageby thrombin, releases 7-amino-4-methylcoumarin, a blue fluorophore thatcan be detected using a fluorescence detector. Alternatives to thrombininclude horseradish peroxidase (HRP), β-galactosidase, and calf alkalinephosphatase (CAP) which can similarly be used to generate a colorimetricor fluorescent signal, and be inhibited by an inhibitory aptamer.

In certain example embodiments, the masking construct may be immobilizedon a solid substrate in an individual discrete volume (defined furtherbelow) and sequesters a single reagent. For example, the reagent may bea bead comprising a dye. When sequestered by the immobilized reagent,the individual beads are too diffuse to generate a detectable signal,but upon release from the masking construct are able to generate adetectable signal, for example by aggregation or simple increase insolution concentration. In certain example embodiments, the immobilizedmasking agent is a RNA-based aptamer that can be cleaved by theactivated effector protein upon detection of a target molecule.

In certain other example embodiments, the masking construct binds to animmobilized reagent in solution thereby blocking the ability of thereagent to bind to a separate labeled binding partner that is free insolution. Thus, upon application of a washing step to a sample, thelabeled binding partner can be washed out of the sample in the absenceof a target molecule. However, if the effector protein is activated, themasking construct is cleaved to a degree sufficient to interfere withthe ability of the masking construct to bind the reagent therebyallowing the labeled binding partner to bind to the immobilized reagent.Thus, the labeled binding partner remains after the wash step indicatingthe presence of the target molecule in the sample. In certain aspects,the masking construct that binds the immobilized reagent is a RNAaptamer. The immobilized reagent may be a protein and the labeledminding partner may be a labeled antibody. Alternatively, theimmobilized reagent may be a streptavidin and the labeled bindingpartner may be labeled biotin. The label on the binding partner used inthe above embodiments may be any detectable label known in the art. Inaddition, other known binding partners may be used in accordance withthe overall design described here.

In certain example embodiments, the masking construct may comprise aribozyme. Ribozymes are RNA molecules having catalytic properties. Asribozymes, both naturally and engineered, comprise or consist of RNA,that may be targeted by the effector proteins disclosed herein. Theribozyme may be selected or engineered to catalyze a reaction thateither generates a negative detectable signal or prevents generation ofa positive control signal. Upon deactivation of the ribozyme by theactivated effector protein molecule the reaction generating a negativecontrols signal or preventing generation of a positive detectable signalis removed, thereby allowing a positive detectable signal to bedetected. In one example embodiment, the ribozyme may catalyze acolorimetric reaction causing a solution to appear as a first color.When the ribozyme is deactivated the solution then turns to a secondcolor, the second color being the detectable positive signal. An exampleof how ribozymes can be used to catalyze a colorimetric reaction aredescribed in Zhao et al. “Signal amplification ofglucosamine-6-phosphate based on ribozyme glmS,” Biosens Bioelectron.2014; 16:337-42, and provide an example of how such a system could bemodified to work in the context of the embodiments disclosed herein.Alternatively, ribozymes, when present can generate cleavage productsof, for example, RNA transcripts. Thus, detection of a positivedetectable signal may comprise detection of non-cleaved RNA transcriptsthat are only generated in the absence of the ribozyme.

In one example embodiment, the masking construct comprises a detectionagent that changes color depending on whether the detection agent isaggregated or dispersed in solution. For example, certain nanoparticles,such as colloidal gold, undergo a visible purple to red color shift asthey move from aggregates to dispersed particles. Accordingly, incertain example embodiments, such detection agents may be held inaggregate by one or more bridge molecules. At least a portion of thebridge molecule comprises RNA. Upon activation of the effector proteinsdisclosed herein, the RNA portion of the bridge molecule is cleavedallowing the detection agent to disperse and resulting in thecorresponding change in color. In certain example embodiments the,bridge molecule is a RNA molecule. In certain example embodiments, thedetection agent is a colloidal metal. The colloidal metal material mayinclude water-insoluble metal particles or metallic compounds dispersedin a liquid, a hydrosol, or a metal sol. The colloidal metal may beselected from the metals in groups IA, IB, IIB and IIIB of the periodictable, as well as the transition metals, especially those of group VIII.Preferred metals include gold, silver, aluminum, ruthenium, zinc, iron,nickel and calcium. Other suitable metals also include the following inall of their various oxidation states: lithium, sodium, magnesium,potassium, scandium, titanium, vanadium, chromium, manganese, cobalt,copper, gallium, strontium, niobium, molybdenum, palladium, indium, tin,tungsten, rhenium, platinum, and gadolinium. The metals are preferablyprovided in ionic form, derived from an appropriate metal compound, forexample the Al³⁺, Ru³⁺, Zn²⁺, Fe³⁺, Ni²⁺ and Ca²⁺ ions.

In certain other example embodiments, the masking construct may comprisean RNA oligonucleotide to which are attached a detectable label and amasking agent of that detectable label. An example of such a detectablelabel/masking agent pair is a fluorophore and a quencher of thefluorophore. Quenching of the fluorophore can occur as a result of theformation of a non-fluorescent complex between the fluorophore andanother fluorophore or non-fluorescent molecule. This mechanism is knownas ground-state complex formation, static quenching, or contactquenching. Accordingly, the RNA oligonucleotide may be designed so thatthe fluorophore and quencher are in sufficient proximity for contactquenching to occur. Fluorophores and their cognate quenchers are knownin the art and can be selected for this purpose by one having ordinaryskill in the art. The particular fluorophore/quencher pair is notcritical in the context of this invention, only that selection of thefluorophore/quencher pairs ensures masking of the fluorophore. Uponactivation of the effector proteins disclosed herein, the RNAoligonucleotide is cleaved thereby severing the proximity between thefluorophore and quencher needed to maintain the contact quenchingeffect. Accordingly, detection of the fluorophore may be used todetermine the presence of a target molecule in a sample.

In one example embodiment, the masking construct may comprise a quantumdot. The quantum dot may have multiple linker molecules attached to thesurface. At least a portion of the linker molecule comprises RNA. Thelinker molecule is attached to the quantum dot at one end and to one ormore quenchers along the length or at terminal ends of the linker suchthat the quenchers are maintained in sufficient proximity for quenchingof the quantum dot to occur. The linker may be branched. As above, thequantum dot/quencher pair is not critical, only that selection of thequantum dot/quencher pair ensures masking of the fluorophore. Quantumdots and their cognate quenchers are known in the art and can beselected for this purpose by one having ordinary skill in the art. Uponactivation of the effector proteins disclosed herein, the RNA portion ofthe linker molecule is cleaved thereby eliminating the proximity betweenthe quantum dot and one or more quenchers needed to maintain thequenching effect. In one embodiment, the quantum dot is streptavidinconjugated, such as Qdot® 625 Streptavidin Conjugate(www.thermofisher.com/order/catalog/product/A10196). RNA are attachedvia biotin linkers and recruit quenching molecules, with the sequence/5Biosg/UCUCGUACGUUC/3IAbRQSp/ (SEQ ID NO: 57) or/5Biosg/UCUCGUACGUUCUCUCGUACGUUC/3IAbRQSp/ (SEQ ID NO: 58) where/5Biosg/ is a biotin tag and /3IAbRQSp/ is an Iowa black quencher. Uponcleavage, the quencher will be released and the quantum dot willfluoresce visibly.

In a similar fashion, fluorescence energy transfer (FRET) may be used togenerate a detectable positive signal. FRET is a non-radiative processby which a photon from an energetically excited fluorophore (i.e. “donorfluorophore”) raises the energy state of an electron in another molecule(i.e. “the acceptor”) to higher vibrational levels of the excitedsinglet state. The donor fluorophore returns to the ground state withoutemitting a fluoresce characteristic of that fluorophore. The acceptorcan be another fluorophore or non-fluorescent molecule. If the acceptoris a fluorophore, the transferred energy is emitted as fluorescencecharacteristic of that fluorophore. If the acceptor is a non-fluorescentmolecule the absorbed energy is loss as heat. Thus, in the context ofthe embodiments disclosed herein, the fluorophore/quencher pair isreplaced with a donor fluorophore/acceptor pair attached to theoligonucleotide molecule. When intact, the masking construct generates afirst signal (negative detectable signal) as detected by thefluorescence or heat emitted from the acceptor. Upon activation of theeffector proteins disclosed herein the RNA oligonucleotide is cleavedand FRET is disrupted such that fluorescence of the donor fluorophore isnow detected (positive detectable signal).

One mode of colorimetric readout for the detection of RNAses is basedupon intercalating dyes, which change their absorbance in response tocleavage of long RNAs to short nucleotides. Several existing dyes withthese properties exist. From Wagner (1983), Pyronine-Y will complex withRNA and form a complex that has an absorbance at 572 nm; cleavage of RNAresults in loss of absorbance and a color change. Greiner-Stoeffele(1996) used methylene blue in a similar fashion, with changes inabsorbance at 688 nm upon RNAse activity.

Another mode of colorimetric readout involves nucleic acid substratesthat change color upon cleavage. Witmer (1991) utilized a syntheticribonucleotide substrate, U-3′-BCIP, that releases a reporter groupafter cleavage, resulting in generation of absorbance at 650 nm.

With respect to general information on CRISPR-Cas Systems, componentsthereof, and delivery of such components, including methods, materials,delivery vehicles, vectors, particles, AAV, and making and usingthereof, including as to amounts and formulations, all useful in thepractice of the instant invention, reference is made to: U.S. Pat. Nos.8,999,641, 8,993,233, 8,945,839, 8,932,814, 8,906,616, 8,895,308,8,889,418, 8,889,356, 8,871,445, 8,865,406, 8,795,965, 8,771,945 and8,697,359; US Patent Publications US 2014-0310830 (U.S. application Ser.No. 14/105,031), US 2014-0287938 A1 (U.S. application Ser. No.14/213,991), US 2014-0273234 A1 (U.S. application Ser. No. 14/293,674),US2014-0273232 A1 (U.S. application Ser. No. 14/290,575), US2014-0273231 (U.S. application Ser. No. 14/259,420), US 2014-0256046 A1(U.S. application Ser. No. 14/226,274), US 2014-0248702 A1 (U.S.application Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. applicationSer. 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Reference is alsomade to U.S. provisional patent application 61/836,123, filed on Jun.17, 2013. Reference is additionally made to U.S. provisional patentapplications 61/835,931, 61/835,936, 61/836,127, 61/836,101, 61/836,080and 61/835,973, each filed Jun. 17, 2013. Further reference is made toU.S. provisional patent applications 61/862,468 and 61/862,355 filed onAug. 5, 2013; 61/871,301 filed on Aug. 28, 2013; 61/960,777 filed onSep. 25, 2013 and 61/961,980 filed on Oct. 28, 2013. Reference is yetfurther made to: PCT Patent applications Nos: PCT/US2014/041803,PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 andPCT/US2014/041806, each filed Jun. 10, 2014; 6/10/14; PCT/US2014/041808filed Jun. 11, 2014; and PCT/US2014/62558 filed Oct. 28, 2014, and U.S.Provisional Patent Applications Ser. Nos. 61/915,150, 61/915,301,61/915,267 and 61/915,260, each filed Dec. 12, 2013; 61/757,972 and61/768,959, filed on Jan. 29, 2013 and Feb. 25, 2013; 61/835,936,61/836,127, 61/836,101, 61/836,080, 61/835,973, and 61/835,931, filedJun. 17, 2013; 62/010,888 and 62/010,879, both filed Jun. 11, 2014;62/010,329 and 62/010,441, each filed Jun. 10, 2014; 61/939,228 and61/939,242, each filed Feb. 12, 2014; 61/980,012, filed Apr. 15, 2014;62/038,358, filed Aug. 17, 2014; 62/054,490, 62/055,484, 62/055,460 and62/055,487, each filed Sep. 25, 2014; and 62/069,243, filed Oct. 27,2014. Reference is also made to U.S. provisional patent applicationsNos. 62/055,484, 62/055,460, and 62/055,487, filed Sep. 25, 2014; U.S.provisional patent application 61/980,012, filed Apr. 15, 2014; and U.S.provisional patent application 61/939,242 filed Feb. 12, 2014. Referenceis made to PCT application designating, inter alia, the United States,application No. PCT/US14/41806, filed Jun. 10, 2014. Reference is madeto U.S. provisional patent application 61/930,214 filed on Jan. 22,2014. Reference is made to U.S. provisional patent applications61/915,251; 61/915,260 and 61/915,267, each filed on Dec. 12, 2013.Reference is made to US provisional patent application U.S. Ser. No.61/980,012 filed Apr. 15, 2014. Reference is made to PCT applicationdesignating, inter alia, the United States, application No.PCT/US14/41806, filed Jun. 10, 2014. Reference is made to U.S.provisional patent application 61/930,214 filed on Jan. 22, 2014.Reference is made to U.S. provisional patent applications 61/915,251;61/915,260 and 61/915,267, each filed on Dec. 12, 2013.

Mention is also made of U.S. application 62/091,455, filed, 12 Dec.2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/096,708, 24Dec. 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. application 62/091,462,12 Dec. 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S.application 62/096,324, 23 Dec. 2014, DEAD GUIDES FOR CRISPRTRANSCRIPTION FACTORS; U.S. application 62/091,456, 12 Dec. 2014,ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S.application 62/091,461, 12 Dec. 2014, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOMEEDITING AS TO HEMATOPOIETIC STEM CELLS (HSCs); U.S. application62/094,903, 19 Dec. 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRANDBREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURESEQUENCING; U.S. application 62/096,761, 24 Dec. 2014, ENGINEERING OFSYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCEMANIPULATION; U.S. application 62/098,059, 30 Dec. 2014, RNA-TARGETINGSYSTEM; U.S. application 62/096,656, 24 Dec. 2014, CRISPR HAVING ORASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. application 62/096,697, 24Dec. 14, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. application62/098,158, 30 Dec. 2014, ENGINEERED CRISPR COMPLEX INSERTIONALTARGETING SYSTEMS; U.S. application 62/151,052, 22 Apr. 15, CELLULARTARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. application62/054,490, 24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS ANDDISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. application62/055,484, 25 Sep. 14, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCEMANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/087,537, 4 Dec. 2014, SYSTEMS, METHODS AND COMPOSITIONSFOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS;U.S. application 62/054,651, 24 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELINGCOMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. application62/067,886, 23 Oct. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OFTHE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OFMULTIPLE CANCER MUTATIONS IN VIVO; U.S. application 62/054,675, 24 Sep.14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. application 62/054,528,24 Sep. 14, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CASSYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS; U.S.application 62/055,454, 25 Sep. 14, DELIVERY, USE AND THERAPEUTICAPPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETINGDISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S.application 62/055,460, 25Sep. 14, MULTIFUNCTIONAL-CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S.application 62/087,475, 4 Dec. 2014, FUNCTIONAL SCREENING WITH OPTIMIZEDFUNCTIONAL CRISPR-CAS SYSTEMS; U.S. application 62/055,487, 25 Sep. 14,FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S.application 62/087,546, 4 Dec. 2014, MULTIFUNCTIONAL CRISPR COMPLEXESAND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S.application 62/098,285, 30 Dec. 2014, CRISPR MEDIATED IN VIVO MODELINGAND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Each of these patents, patent publications, and applications, and alldocuments cited therein or during their prosecution (“appln. citeddocuments”) and all documents cited or referenced in the appln. citeddocuments, together with any instructions, descriptions, productspecifications, and product sheets for any products mentioned therein orin any document therein and incorporated by reference herein, are herebyincorporated herein by reference, and may be employed in the practice ofthe invention. All documents (e.g., these patents, patent publicationsand applications and the appln. cited documents) are incorporated hereinby reference to the same extent as if each individual document wasspecifically and individually indicated to be incorporated by reference.

Also with respect to general information on CRISPR-Cas Systems, mentionis made of the following (also hereby incorporated herein by reference):

-   Multiplex genome engineering using CRISPR/Cas systems. Cong, L.,    Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D.,    Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. Science February    15; 339(6121):819-23 (2013);-   RNA-guided editing of bacterial genomes using CRISPR-Cas systems.    Jiang W., Bikard D., Cox D., Zhang F, Marraffini L A. Nat Biotechnol    March; 31(3):233-9 (2013);-   One-Step Generation of Mice Carrying Mutations in Multiple Genes by    CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila    C S., Dawlaty M M., Cheng A W., Zhang F., Jaenisch R. Cell May 9;    153(4):910-8 (2013);-   Optical control of mammalian endogenous transcription and epigenetic    states. Konermann S, Brigham M D, Trevino A E, Hsu P D, Heidenreich    M, Cong L, Platt R J, Scott D A, Church G M, Zhang F. Nature. August    22; 500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug. 23    (2013);-   Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing    Specificity. Ran, F A., Hsu, P D., Lin, C Y., Gootenberg, J S.,    Konermann, S., Trevino, A E., Scott, D A., Inoue, A., Matoba, S.,    Zhang, Y., & Zhang, F. Cell August 28. pii: S0092-8674(13)01015-5    (2013-A);-   DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P.,    Scott, D., Weinstein, J., Ran, F A., Konermann, S., Agarwala, V.,    Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T J., Marraffini, L    A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);-   Genome engineering using the CRISPR-Cas9 system. Ran, F A., Hsu, P    D., Wright, J., Agarwala, V., Scott, D A., Zhang, F. Nature    Protocols November; 8(11):2281-308 (2013-B);-   Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem,    O., Sanjana, N E., Hartenian, E., Shi, X., Scott, D A., Mikkelson,    T., Heckl, D., Ebert, B L., Root, D E., Doench, J G., Zhang, F.    Science Dec. 12, 2013. [Epub ahead of print];-   Crystal structure of cas9 in complex with guide RNA and target DNA.    Nishimasu, H., Ran, F A., Hsu, P D., Konermann, S., Shehata, S I.,    Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell February 27,    156(5):935-49 (2014);-   Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian    cells. Wu X., Scott D A., Kriz A J., Chiu A C., Hsu P D., Dadon D    B., Cheng A W., Trevino A E., Konermann S., Chen S., Jaenisch R.,    Zhang F., Sharp P A. Nat Biotechnol. April 20. doi: 10.1038/nbt.2889    (2014);-   CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling.    Platt R J, Chen S, Zhou Y, Yim M J, Swiech L, Kempton H R, Dahlman J    E, Parnas O, Eisenhaure T M, Jovanovic M, Graham D B, Jhunjhunwala    S, Heidenreich M, Xavier R J, Langer R, Anderson D G, Hacohen N,    Regev A, Feng G, Sharp P A, Zhang F. Cell 159(2): 440-455 DOI:    10.1016/j.cell.2014.09.014 (2014);-   Development and Applications of CRISPR-Cas9 for Genome Engineering,    Hsu P D, Lander E S, Zhang F., Cell. June 5; 157(6):1262-78 (2014);-   Genetic screens in human cells using the CRISPR/Cas9 system, Wang T,    Wei J J, Sabatini D M, Lander E S., Science. January 3; 343(6166):    80-84. doi:10.1126/science.1246981 (2014);-   Rational design of highly active sgRNAs for CRISPR-Cas9-mediated    gene inactivation, Doench J G, Hartenian E, Graham D B, Tothova Z,    Hegde M, Smith I, Sullender M, Ebert B L, Xavier R J, Root D E.,    (published online 3 Sep. 2014) Nat Biotechnol. December;    32(12):1262-7 (2014);-   In vivo interrogation of gene function in the mammalian brain using    CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y,    Trombetta J, Sur M, Zhang F., (published online 19 Oct. 2014) Nat    Biotechnol. January; 33(1):102-6 (2015);-   Genome-scale transcriptional activation by an engineered CRISPR-Cas9    complex, Konermann S, Brigham M D, Trevino A E, Joung J, Abudayyeh    OO, Barcena C, Hsu P D, Habib N, Gootenberg J S, Nishimasu H, Nureki    O, Zhang F., Nature. January 29; 517(7536):583-8 (2015);-   A split-Cas9 architecture for inducible genome editing and    transcription modulation, Zetsche B, Volz S E, Zhang F., (published    online 2 Feb. 2015) Nat Biotechnol. February; 33(2):139-42 (2015);-   Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and    Metastasis, Chen S, Sanjana N E, Zheng K, Shalem 0, Lee K, Shi X,    Scott D A, Song J, Pan J Q, Weissleder R, Lee H, Zhang F, Sharp P A.    Cell 160, 1246-1260, Mar. 12, 2015 (multiplex screen in mouse), and-   In vivo genome editing using Staphylococcus aureus Cas9, Ran F A,    Cong L, Yan W X, Scott D A, Gootenberg J S, Kriz A J, Zetsche B,    Shalem 0, Wu X, Makarova K S, Koonin E V, Sharp P A, Zhang F.,    (published online 1 Apr. 2015), Nature. April 9; 520(7546):186-91    (2015);-   Shalem et al., “High-throughput functional genomics using    CRISPR-Cas9,” Nature Reviews Genetics 16, 299-311 (May 2015);-   Xu et al., “Sequence determinants of improved CRISPR sgRNA design,”    Genome Research 25, 1147-1157 (August 2015);-   Parnas et al., “A Genome-wide CRISPR Screen in Primary Immune Cells    to Dissect Regulatory Networks,” Cell 162, 675-686 (Jul. 30, 2015)′-   Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently    suppresses hepatitis B virus,” Scientific Reports 5:10833. doi:    10.1038/srep10833 (Jun. 2, 2015);-   Nishimasu et al., “Crystal Structure of Staphylococcus aureus Cas9,”    Cell 162, 1113-1126 (Aug. 27, 2015);-   Zetsche et al. (2015), “Cpf1 is a single RNA-guided endonuclease of    a class 2 CRISPR-Cas system,” Cell 163, 759-771 (Oct. 22, 2015) doi:    10.1016/j.cell.2015.09.038. Epub Sep. 25, 2015;-   Shmakov et al. (2015), “Discovery and Functional Characterization of    Diverse Class 2 CRISPR-Cas Systems,” Molecular Cell 60, 385-397    (Nov. 5, 2015) doi: 10.1016/j.molcel.2015.10.008. Epub Oct. 22,    2015;-   Dahlman et al., “Orthogonal gene control with a catalytically active    Cas9 nuclease,” Nature Biotechnology 33, 1159-1161 (November, 2015);-   Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,”    bioRxiv 091611; doi: http://dx.doi.org/10.1101/091611 Epub Dec. 4,    2016;-   Smargon et al. (2017), “Cas13b Is a Type VI-B CRISPR-Associated    RNA-Guided rNase Differentially Regulated by Accessory Proteins    Csx27 and Csx28,” Molecular Cell 65, 618-630 (Feb. 16, 2017) doi:    10.1016/j.molcel.2016.12.023. Epub Jan. 5, 2017;    each of which is incorporated herein by reference, may be considered    in the practice of the instant invention, and discussed briefly    below:-   Cong et al. engineered type II CRISPR-Cas systems for use in    eukaryotic cells based on both Streptococcus thermophilus Cas9 and    also Streptococcus pyogenes Cas9 and demonstrated that Cas9    nucleases can be directed by short RNAs to induce precise cleavage    of DNA in human and mouse cells. Their study further showed that    Cas9 as converted into a nicking enzyme can be used to facilitate    homology-directed repair in eukaryotic cells with minimal mutagenic    activity. Additionally, their study demonstrated that multiple guide    sequences can be encoded into a single CRISPR array to enable    simultaneous editing of several at endogenous genomic loci sites    within the mammalian genome, demonstrating easy programmability and    wide applicability of the RNA-guided nuclease technology. This    ability to use RNA to program sequence specific DNA cleavage in    cells defined a new class of genome engineering tools. These studies    further showed that other CRISPR loci are likely to be    transplantable into mammalian cells and can also mediate mammalian    genome cleavage. Importantly, it can be envisaged that several    aspects of the CRISPR-Cas system can be further improved to increase    its efficiency and versatility.-   Jiang et al. used the clustered, regularly interspaced, short    palindromic repeats (CRISPR)-associated Cas9 endonuclease complexed    with dual-RNAs to introduce precise mutations in the genomes of    Streptococcus pneumoniae and Escherichia coli. The approach relied    on dual-RNA:Cas9-directed cleavage at the targeted genomic site to    kill unmutated cells and circumvents the need for selectable markers    or counter-selection systems. The study reported reprogramming    dual-RNA:Cas9 specificity by changing the sequence of short CRISPR    RNA (crRNA) to make single- and multinucleotide changes carried on    editing templates. The study showed that simultaneous use of two    crRNAs enabled multiplex mutagenesis. Furthermore, when the approach    was used in combination with recombineering, in S. pneumoniae,    nearly 100% of cells that were recovered using the described    approach contained the desired mutation, and in E. coli, 65% that    were recovered contained the mutation.-   Wang et al. (2013) used the CRISPR/Cas system for the one-step    generation of mice carrying mutations in multiple genes which were    traditionally generated in multiple steps by sequential    recombination in embryonic stem cells and/or time-consuming    intercrossing of mice with a single mutation. The CRISPR/Cas system    will greatly accelerate the in vivo study of functionally redundant    genes and of epistatic gene interactions.-   Konermann et al. (2013) addressed the need in the art for versatile    and robust technologies that enable optical and chemical modulation    of DNA-binding domains based CRISPR Cas9 enzyme and also    Transcriptional Activator Like Effectors.-   Ran et al. (2013-A) described an approach that combined a Cas9    nickase mutant with paired guide RNAs to introduce targeted    double-strand breaks. This addresses the issue of the Cas9 nuclease    from the microbial CRISPR-Cas system being targeted to specific    genomic loci by a guide sequence, which can tolerate certain    mismatches to the DNA target and thereby promote undesired    off-target mutagenesis. Because individual nicks in the genome are    repaired with high fidelity, simultaneous nicking via appropriately    offset guide RNAs is required for double-stranded breaks and extends    the number of specifically recognized bases for target cleavage. The    authors demonstrated that using paired nicking can reduce off-target    activity by 50- to 1,500-fold in cell lines and to facilitate gene    knockout in mouse zygotes without sacrificing on-target cleavage    efficiency. This versatile strategy enables a wide variety of genome    editing applications that require high specificity.-   Hsu et al. (2013) characterized SpCas9 targeting specificity in    human cells to inform the selection of target sites and avoid    off-target effects. The study evaluated >700 guide RNA variants and    SpCas9-induced indel mutation levels at >100 predicted genomic    off-target loci in 293T and 293FT cells. The authors that SpCas9    tolerates mismatches between guide RNA and target DNA at different    positions in a sequence-dependent manner, sensitive to the number,    position and distribution of mismatches. The authors further showed    that SpCas9-mediated cleavage is unaffected by DNA methylation and    that the dosage of SpCas9 and sgRNA can be titrated to minimize    off-target modification. Additionally, to facilitate mammalian    genome engineering applications, the authors reported providing a    web-based software tool to guide the selection and validation of    target sequences as well as off-target analyses.-   Ran et al. (2013-B) described a set of tools for Cas9-mediated    genome editing via non-homologous end joining (NHEJ) or    homology-directed repair (HDR) in mammalian cells, as well as    generation of modified cell lines for downstream functional studies.    To minimize off-target cleavage, the authors further described a    double-nicking strategy using the Cas9 nickase mutant with paired    guide RNAs. The protocol provided by the authors experimentally    derived guidelines for the selection of target sites, evaluation of    cleavage efficiency and analysis of off-target activity. The studies    showed that beginning with target design, gene modifications can be    achieved within as little as 1-2 weeks, and modified clonal cell    lines can be derived within 2-3 weeks.-   Shalem et al. described a new way to interrogate gene function on a    genome-wide scale. Their studies showed that delivery of a    genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080    genes with 64,751 unique guide sequences enabled both negative and    positive selection screening in human cells. First, the authors    showed use of the GeCKO library to identify genes essential for cell    viability in cancer and pluripotent stem cells. Next, in a melanoma    model, the authors screened for genes whose loss is involved in    resistance to vemurafenib, a therapeutic that inhibits mutant    protein kinase BRAF. Their studies showed that the highest-ranking    candidates included previously validated genes NF1 and MED12 as well    as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a    high level of consistency between independent guide RNAs targeting    the same gene and a high rate of hit confirmation, and thus    demonstrated the promise of genome-scale screening with Cas9.-   Nishimasu et al. reported the crystal structure of Streptococcus    pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A°    resolution. The structure revealed a bilobed architecture composed    of target recognition and nuclease lobes, accommodating the    sgRNA:DNA heteroduplex in a positively charged groove at their    interface. Whereas the recognition lobe is essential for binding    sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease    domains, which are properly positioned for cleavage of the    complementary and non-complementary strands of the target DNA,    respectively. The nuclease lobe also contains a carboxyl-terminal    domain responsible for the interaction with the protospacer adjacent    motif (PAM). This high-resolution structure and accompanying    functional analyses have revealed the molecular mechanism of    RNA-guided DNA targeting by Cas9, thus paving the way for the    rational design of new, versatile genome-editing technologies.-   Wu et al. mapped genome-wide binding sites of a catalytically    inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single    guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). The    authors showed that each of the four sgRNAs tested targets dCas9 to    between tens and thousands of genomic sites, frequently    characterized by a 5-nucleotide seed region in the sgRNA and an NGG    protospacer adjacent motif (PAM). Chromatin inaccessibility    decreases dCas9 binding to other sites with matching seed sequences;    thus 70% of off-target sites are associated with genes. The authors    showed that targeted sequencing of 295 dCas9 binding sites in mESCs    transfected with catalytically active Cas9 identified only one site    mutated above background levels. The authors proposed a two-state    model for Cas9 binding and cleavage, in which a seed match triggers    binding but extensive pairing with target DNA is required for    cleavage.-   Platt et al. established a Cre-dependent Cas9 knockin mouse. The    authors demonstrated in vivo as well as ex vivo genome editing using    adeno-associated virus (AAV)-, lentivirus-, or particle-mediated    delivery of guide RNA in neurons, immune cells, and endothelial    cells.-   Hsu et al. (2014) is a review article that discusses generally    CRISPR-Cas9 history from yogurt to genome editing, including genetic    screening of cells.-   Wang et al. (2014) relates to a pooled, loss-of-function genetic    screening approach suitable for both positive and negative selection    that uses a genome-scale lentiviral single guide RNA (sgRNA)    library.-   Doench et al. created a pool of sgRNAs, tiling across all possible    target sites of a panel of six endogenous mouse and three endogenous    human genes and quantitatively assessed their ability to produce    null alleles of their target gene by antibody staining and flow    cytometry. The authors showed that optimization of the PAM improved    activity and also provided an on-line tool for designing sgRNAs.-   Swiech et al. demonstrate that AAV-mediated SpCas9 genome editing    can enable reverse genetic studies of gene function in the brain.-   Konermann et al. (2015) discusses the ability to attach multiple    effector domains, e.g., transcriptional activator, functional and    epigenomic regulators at appropriate positions on the guide such as    stem or tetraloop with and without linkers.-   Zetsche et al. demonstrates that the Cas9 enzyme can be split into    two and hence the assembly of Cas9 for activation can be controlled.-   Chen et al. relates to multiplex screening by demonstrating that a    genome-wide in vivo CRISPR-Cas9 screen in mice reveals genes    regulating lung metastasis.-   Ran et al. (2015) relates to SaCas9 and its ability to edit genomes    and demonstrates that one cannot extrapolate from biochemical    assays. Shalem et al. (2015) described ways in which catalytically    inactive Cas9 (dCas9) fusions are used to synthetically repress    (CRISPRi) or activate (CRISPRa) expression, showing advances using    Cas9 for genome-scale screens, including arrayed and pooled screens,    knockout approaches that inactivate genomic loci and strategies that    modulate transcriptional activity.-   Shalem et al. (2015) described ways in which catalytically inactive    Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or    activate (CRISPRa) expression, showing advances using Cas9 for    genome-scale screens, including arrayed and pooled screens, knockout    approaches that inactivate genomic loci and strategies that modulate    transcriptional activity.-   Xu et al. (2015) assessed the DNA sequence features that contribute    to single guide RNA (sgRNA) efficiency in CRISPR-based screens. The    authors explored efficiency of CRISPR/Cas9 knockout and nucleotide    preference at the cleavage site. The authors also found that the    sequence preference for CRISPRi/a is substantially different from    that for CRISPR/Cas9 knockout.-   Parnas et al. (2015) introduced genome-wide pooled CRISPR-Cas9    libraries into dendritic cells (DCs) to identify genes that control    the induction of tumor necrosis factor (Tnf) by bacterial    lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and    previously unknown candidates were identified and classified into    three functional modules with distinct effects on the canonical    responses to LPS.-   Ramanan et al (2015) demonstrated cleavage of viral episomal DNA    (cccDNA) in infected cells. The HBV genome exists in the nuclei of    infected hepatocytes as a 3.2kb double-stranded episomal DNA species    called covalently closed circular DNA (cccDNA), which is a key    component in the HBV life cycle whose replication is not inhibited    by current therapies. The authors showed that sgRNAs specifically    targeting highly conserved regions of HBV robustly suppresses viral    replication and depleted cccDNA.-   Nishimasu et al. (2015) reported the crystal structures of SaCas9 in    complex with a single guide RNA (sgRNA) and its double-stranded DNA    targets, containing the “-TTGAAT-” PAM and the “-TTGGGT-” PAM. A    structural comparison of SaCas9 with SpCas9 highlighted both    structural conservation and divergence, explaining their distinct    PAM specificities and orthologous sgRNA recognition.-   Zetsche et al. (2015) reported the characterization of Cpf1, a    putative class 2 CRISPR effector. It was demonstrated that Cpf1    mediates robust DNA interference with features distinct from Cas9.    Identifying this mechanism of interference broadens our    understanding of CRISPR-Cas systems and advances their genome    editing applications.-   Shmakov et al. (2015) reported the characterization of three    distinct Class 2 CRISPR-Cas systems. The effectors of two of the    identified systems, C2c1 and C2c3, contain RuvC like endonuclease    domains distantly related to Cpf1. The third system, C2c2, contains    an effector with two predicted HEPN rNase domains.-   Gao et al. (2016) reported using a structure-guided saturation    mutagenesis screen to increase the targeting range of Cpf1. AsCpf1    variants were engineered with the mutations S542R/K607R and    S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and    TATV PAMs, respectively, with enhanced activities in vitro and in    human cells.

Also, “Dimeric CRISPR RNA-guided FokI nucleases for highly specificgenome editing”, Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter,Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin,Martin J. Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77(2014), relates to dimeric RNA-guided FokI Nucleases that recognizeextended sequences and can edit endogenous genes with high efficienciesin human cells.

In addition, mention is made of PCT application PCT/US14/70057, entitled“DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMSAND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLEDELIVERY COMPONENTS (claiming priority from one or more or all of USprovisional patent applications: 62/054,490, filed Sep. 24, 2014;62/010,441, filed Jun. 10, 2014; and 61/915,118, 61/915,215 and61/915,148, each filed on Dec. 12, 2013) (“the Particle Delivery PCT”),incorporated herein by reference, with respect to a method of preparingan sgRNA-and-Cas9 protein containing particle comprising admixing amixture comprising an sgRNA and Cas9 protein (and optionally HDRtemplate) with a mixture comprising or consisting essentially of orconsisting of surfactant, phospholipid, biodegradable polymer,lipoprotein and alcohol; and particles from such a process. For example,wherein Cas9 protein and sgRNA were mixed together at a suitable, e.g.,3:1 to 1:3 or 2:1 to 1:2 or 1:1 molar ratio, at a suitable temperature,e.g., 15-30° C., e.g., 20-25° C., e.g., room temperature, for a suitabletime, e.g., 15-45, such as 30 minutes, advantageously in sterile,nuclease free buffer, e.g., 1×PBS. Separately, particle components suchas or comprising: a surfactant, e.g., cationic lipid, e.g.,1,2-dioleoyl-3-trimethylammonium-propane (DOTAP); phospholipid, e.g.,dimyristoylphosphatidylcholine (DMPC); biodegradable polymer, such as anethylene-glycol polymer or PEG, and a lipoprotein, such as a low-densitylipoprotein, e.g., cholesterol were dissolved in an alcohol,advantageously a C₁₋₆ alkyl alcohol, such as methanol, ethanol,isopropanol, e.g., 100% ethanol. The two solutions were mixed togetherto form particles containing the Cas9-sgRNA complexes. Accordingly,sgRNA may be pre-complexed with the Cas9 protein, before formulating theentire complex in a particle. Formulations may be made with a differentmolar ratio of different components known to promote delivery of nucleicacids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol) For example DOTAP:DMPC:PEG:CholesterolMolar Ratios may be DOTAP 100, DMPC 0, PEG 0, Cholesterol 0; or DOTAP90, DMPC 0, PEG 10, Cholesterol 0; or DOTAP 90, DMPC 0, PEG 5,Cholesterol 5, DOTAP 100, DMPC 0, PEG 0, Cholesterol 0. That applicationaccordingly comprehends admixing sgRNA, Cas9 protein and components thatform a particle; as well as particles from such admixing. Aspects of theinstant invention can involve particles; for example, particles using aprocess analogous to that of the Particle Delivery PCT, e.g., byadmixing a mixture comprising sgRNA and/or Cas9 as in the instantinvention and components that form a particle, e.g., as in the ParticleDelivery PCT, to form a particle and particles from such admixing (or,of course, other particles involving sgRNA and/or Cas9 as in the instantinvention).

EXAMPLES Example 1: Characterization of Cas13a Family

Applicants comprehensively evaluated fifteen Cas13a orthologs for PFSpreference and activity (FIG. 57A) using an ampicillin resistancebacterial assay (FIG. 3A). Briefly, Cas13a is programmed to target a 5′stretch of sequence on the P-lactamase transcript flanked by randomizedPFS nucleotides. Cas13a cleavage activity results in death of bacteriaunder ampicillin selection and PFS depletion is subsequently analyzed bynext generation sequencing. In order to allow for quantitativecomparisons between orthologs, Applicants cloned each Cas13a orthologunder a pLac promoter along with a single-spacer CRISPR array nearbyunder expression of the pJ23119 small RNA promoter. Afterco-transformation of the PFS plasmid library with each of the Cas13aortholog plasmids in E. coli, Applicants measured bacteria survival viatransformant counting and found that the Cas13a ortholog fromLeptotrichia wadei (LwaCas13a) was most active, followed by Leptotrichiashahii Cas13a (LshCas13a) (FIG. 3C, D). Next generation sequencinganalysis of the PFS distributions from LwaCas13a and LshCas13a screensrevealed that most LwCas13a PFS sequences were depleted (FIGS. 3G and57B, C), suggesting robust LwCas13a RNA cleavage activity. Indeed, motifanalysis of the depleted PFS sequences at varying thresholds revealedthe expected 3′ H motif of LshCas13a, but no significant PFS motif forLwCas13a (FIGS. 3H, 56D, E). In the recent development of LwCas13a fornucleic acid detection, Applicants found LwCas13a to be more active thanLshCas13a and found a weak 3′ H PFS by biochemical characterization.LwCas13a was found to be most active of the fifteen Cas13a orthologstested and to have no PFS in bacteria.

Example 2: Expression of C2c2 in Eukaryotic Cells

The following C2c2 orthologues were codon optimized for expression inmammalian cells.

C2c2 orthologue Code Multi Letter Leptotrichia shahii C2-2 Lsh L wadeiF0279 (Lw2) C2-3 Lw2 Listeria seeliged C2-4 Lse Lachnospiraceaebacterium MA2020 C2-5 LbM Lachnospiraceae bacterium NK4A179 C2-6 LbNK179[Clostridium] aminophilum DSM 10710 C2-7 Ca Carnobacterium gallinarumDSM 4847 C2-8 Cg Carnobacterium gallinarum DSM 4847 C2-9 Cg2Paludibacter propionicigenes WB4 C2-10 Pp Listeria weihenstephanensisFSL R9-0317 C2-11 Lwei Listeriaceae bacterium FSL M6-0635 C2-12 LbFSLLeptotrichia wadei F0279 C2-13 Lw Rhodobacter capsulatus SB 1003 C2-14Rc Rhodobacter capsulatus R121 C2-15 Rc Rhodobacter capsulatus DE442C2-16 Rc

The protein sequences of the above species are listed in the Tablebelow.

c2c2-5 1 Lachnospiraceae MQISKVNHKHVAVGQKDRERITGFIYNDPVGDEKSLEDVVAKRAbacterium NDTKVLFNVFNTKDLYDSQESDKSEKDKEIISKGAKFVAKSFNSAI MA2020TILKKQNKIYSTLTSQQVIKELKDKFGGARIYDDDIEEALTETLKKSFRKENVRNSIKVLIENAAGIRSSLSKDEEELIQEYFVKQLVEEYTKTKLQKNVVKSIKNQNMVIQPDSDSQVLSLSESRREKQSSAVSSDTLVNCKEKDVLKAFLTDYAVLDEDERNSLLWKLRNLVNLYFYGSESIRDYSYTKEKSVWKEHDEQKANKTLFIDEICHITKIGKNGKEQKVLDYEENRSRCRKQNINYYRSALNYAKNNTSGIFENEDSNHFWHLIENEVERLYNGIENGEEFKFETGYISEKVWKAVINHLSIKYIALGKAVYNYAMKELSSPGDIEPGKIDDSYINGITSFDYEIIKAEESLQRDISMNVVFATNYLACATVDTDKDFLLFSKEDIRSCTKKDGNLCKNIMQFWGGYSTWKNFCEEYLKDDKDALELLYSLKSMLYSMRNSSFHFSTENVDNGSWDTELIGKLFEEDCNRAARIEKEKFYNNNLHMFYSSSLLEKVLERIYSSHHERASQVPSFNRVFVRKNFPSSLSEQRITPKFTDSKDEQIWQSAVYYLCKEIYYNDFLQSKEAYKLFREGVKNLDKNDINNQKAADSFKQAVVYYGKAIGNATLSQVCQAIMTEYNRQNNDGLKKKSAYAEKQNSNKYKHYPLFLKQVLQSAFWEYLDENKEIYGFISAQIHKSNVEIKAEDFIANYSSQQYKKLVDKVKKTPELQKWYTLGRLINPRQANQFLGSIRNYVQFVKDIQRRAKENGNPIRNYYEVLESDSIIKILEMCTKLNGTTSNDIHDYFRDEDEYAEYISQFVNFGDVHSGAALNAFCNSESEGKKNGIYYDGINPIVNRNWVLCKLYGSPDLISKIISRVNENMIHDFHKQEDLIREYQIKGICSNKKEQQDLRTFQVLKNRVELRDIVEYSEIINELYGQLIKWCYLRERDLMYFQLGFHYLCLNNASSKEADYIKINVDDRNISGAILYQIAAMYINGLPVYYKKDDMYVALKSGKKASDELNSNEQTSKKINYFLKYGNNILGDKKDQLYLAGLELFENVAEHENIIIFRNEIDHFHYFYDRDRSMLDLYSEVFDRFFTYDMKLRKNVVNMLYNILLDHNIVSSFVFETGEKKVGRGDSEVIKPSAKIRLRANNGVSSDVFTYKVGSKDELKIATLPAKNEEFLLNVARLIYYPDMEAVSENMVREGVVKVEKSNDKKGKISRGSNTRSSNQSKYNNKSKNRMNYSMGSIFEKMDLKFD (SEQ ID NO: 59) c2c2-6 2 LachnospiraceaeMKISKVREENRGAKLTVNAKTAVVSENRSQEGILYNDPSRYGKSR bacteriumKNDEDRDRYIESRLKSSGKLYRIFNEDKNKRETDELQWFLSEIVKK NK4A179INRRNGLVLSDMLSVDDRAFEKAFEKYAELSYTNRRNKVSGSPAFETCGVDAATAERLKGIISETNFINRIKNNIDNKVSEDIIDRIIAKYLKKSLCRERVKRGLKKLLMNAFDLPYSDPDIDVQRDFIDYVLEDFYHVRAKSQVSRSIKNMNMPVQPEGDGKFAITVSKGGTESGNKRSAEKEAFKKFLSDYASLDERVRDDMLRRMRRLVVLYFYGSDDSKLSDVNEKFDVWEDHAARRVDNREFIKLPLENKLANGKTDKDAERIRKNTVKELYRNQNIGCYRQAVKAVEEDNNGRYFDDKMLNMFFIHRIEYGVEKIYANLKQVTEFKARTGYLSEKIWKDLINYISIKYIAMGKAVYNYAMDELNASDKKEIELGKISEEYLSGISSFDYELIKAEEMLQRETAVYVAFAARHLSSQTVELDSENSDFLLLKPKGTMDKNDKNKLASNNILNFLKDKETLRDTILQYFGGHSLWTDFPFDKYLAGGKDDVDFLTDLKDVIYSMRNDSFHYATENHNNGKWNKELISAMFEHETERMTVVMKDKFYSNNLPMFYKNDDLKKLLIDLYKDNVERASQVPSFNKVFVRKNFPALVRDKDNLGIELDLKADADKGENELKFYNALYYMFKEIYYNAFLNDKNVRERFITKATKVADNYDRNKERNLKDRIKSAGSDEKKKLREQLQNYIAENDFGQRIKNIVQVNPDYTLAQICQLIMTEYNQQNNGCMQKKSAARKDINKDSYQHYKMLLLVNLRKAFLEFIKENYAFVLKPYKHDLCDKADFVPDFAKYVKPYAGLISRVAGSSELQKWYIVSRFLSPAQANHMLGFLHSYKQYVWDIYRRASETGTEINHSIAEDKIAGVDITDVDAVIDLSVKLCGTISSEISDYFKDDEVYAEYISSYLDFEYDGGNYKDSLNRFCNSDAVNDQKVALYYDGEHPKLNRNIILSKLYGERRFLEKITDRVSRSDIVEYYKLKKETSQYQTKGIFDSEDEQKNIKKFQEMKNIVEFRDLMDYSEIADELQGQLINWIYLRERDLMNFQLGYHYACLNNDSNKQATYVTLDYQGKKNRKINGAILYQICAMYINGLPLYYVDKDSSEWTVSDGKESTGAKIGEFYRYAKSFENTSDCYASGLEIFENISEHDNITELRNYIEHFRWSSFDRSFLGIYSEVFDRFFTYDLKYRKNVPTILYNILLQHFVNVRFEFVSGKKMIGIDKKDRKIAKEKECARITIREKNGVYSEQFTYKLKNGTVYVDARDKRYLQSIIRLLFYPEKVNMDEMIEVKEKKKPSDNNTGKGYSKRDRQQDRKEYDKYKEKKKKEGNFLSGMGGNINWDEINAQLKN (SEQ ID NO: 60) c2c2-7 3[Clostridium] MKFSKVDHTRSAVGIQKATDSVHGMLYTDPKKQEVNDLDKRFDQ aminophilumLNVKAKRLYNVFNQSKAEEDDDEKRFGKVVKKLNRELKDLLFHR DSMEVSRYNSIGNAKYNYYGIKSNPEEIVSNLGMVESLKGERDPQKVIS 10710KLLLYYLRKGLKPGTDGLRMILEASCGLRKLSGDEKELKVFLQTLDEDFEKKTFKKNLIRSIENQNMAVQPSNEGDPIIGITQGRFNSQKNEEKSAIERMMSMYADLNEDHREDVLRKLRRLNVLYFNVDTEKTEEPTLPGEVDTNPVFEVWHDHEKGKENDRQFATFAKILTEDRETRKKEKLAVKEALNDLKSAIRDHNIMAYRCSIKVTEQDKDGLFFEDQRINRFWIHHIESAVERILASINPEKLYKLRIGYLGEKVWKDLLNYLSIKYIAVGKAVFHFAMEDLGKTGQDIELGKLSNSVSGGLTSFDYEQIRADETLQRQLSVEVAFAANNLFRAWGQTGKKIEQSKSEENEEDFLLWKAEKIAESIKKEGEGNTLKSILQFFGGASSWDLNHFCAAYGNESSALGYETKFADDLRKAIYSLRNETFHFTTLNKGSFDWNAKLIGDMFSHEAATGIAVERTRFYSNNLPMFYRESDLKRIMDHLYNTYHPRASQVPSFNSVFVRKNFRLFLSNTLNTNTSFDTEVYQKWESGVYYLFKEIYYNSFLPSGDAHHLFFEGLRRIRKEADNLPIVGKEAKKRNAVQDFGRRCDELKNLSLSAICQMIMTEYNEQNNGNRKVKSTREDKRKPDIFQHYKMLLLRTLQEAFAIYIRREEFKFIFDLPKTLYVMKPVEEFLPNWKSGMFDSLVERVKQSPDLQRWYVLCKFLNGRLLNQLSGVIRSYIQFAGDIQRRAKANHNRLYMDNTQRVEYYSNVLEVVDFCIKGTSRFSNVFSDYFRDEDAYADYLDNYLQFKDEKIAEVSSFAALKTFCNEEEVKAGIYMDGENPVMQRNIVMAKLFGPDEVLKNVVPKVTREEIEEYYQLEKQIAPYRQNGYCKSEEDQKKLLRFQRIKNRVEFQTITEFSEIINELLGQLISWSFLRERDLLYFQLGFHYLCLHNDTEKPAEYKEISREDGTVIRNAILHQVAAMYVGGLPVYTLADKKLAAFEKGEADCKLSISKDTAGAGKKIKDFFRYSKYVLIKDRMLTDQNQKYTIYLAGLELFENTDEHDNITDVRKYVDHFKYYATSDENAMSILDLYSEIHDRFFTYDMKYQKNVANMLENILLRHFVLIRPEFFTGSKKVGEGKKITCKARAQIEIAENGMRSEDFTYKLSDGKKNISTCMIAARDQKYLNTVARLLYYPHEAKKSIVDTREKKNNKKTNRGDGTFNKQKGTARKEKDNGPREFNDTGFSNTPFAGFDPFRNS (SEQ ID NO: 61) c2c2-8 5 CarnobacteriumMRITKVKIKLDNKLYQVTMQKEEKYGTLKLNEESRKSTAEILRLK gallinarumKASFNKSFHSKTINSQKENKNATIKKNGDYISQIFEKLVGVDTNKNI DSM 4847RKPKMSLTDLKDLPKKDLALFIKRKFKNDDIVEIKNLDLISLFYNALQKVPGEHFTDESWADFCQEMMPYREYKNKFIERKIILLANSIEQNKGFSINPETFSKRKRVLHQWAIEVQERGDFSILDEKLSKLAEIYNFKKMCKRVQDELNDLEKSMKKGKNPEKEKEAYKKQKNFKIKTIWKDYPYKTHIGLIEKIKENEELNQFNIEIGKYFEHYFPIKKERCTEDEPYYLNSETIATTVNYQLKNALISYLMQIGKYKQFGLENQVLDSKKLQEIGIYEGFQTKFMDACVFATSSLKNIIEPMRSGDILGKREFKEAIATSSFVNYHHFFPYFPFELKGMKDRESELIPFGEQTEAKQMQNIWALRGSVQQIRNEIFHSFDKNQKFNLPQLDKSNFEFDASENSTGKSQSYIETDYKFLFEAEKNQLEQFFIERIKSSGALEYYPLKSLEKLFAKKEMKFSLGSQVVAFAPSYKKLVKKGHSYQTATEGTANYLGLSYYNRYELKEESFQAQYYLLKLIYQYVFLPNFSQGNSPAFRETVKAILRINKDEARKKMKKNKKFLRKYAFEQVREMEFKETPDQYMSYLQSEMREEKVRKAEKNDKGFEKNITMNFEKLLMQIFVKGFDVFLTTFAGKELLLSSEEKVIKETEISLSKKINEREKTLKASIQVEHQLVATNSAISYWLFCKLLDSRHLNELRNEMIKFKQSRIKFNHTQHAELIQNLLPIVELTILSNDYDEKNDSQNVDVSAYFEDKSLYETAPYVQTDDRTRVSFRPILKLEKYHTKSLIEALLKDNPQFRVAATDIQEWMHKREEIGELVEKRKNLHTEWAEGQQTLGAEKREEYRDYCKKIDRFNWKANKVTLTYLSQLHYLITDLLGRMVGFSALFERDLVYFSRSFSELGGETYHISDYKNLSGVLRLNAEVKPIKIKNIKVIDNEENPYKGNEPEVKPFLDRLHAYLENVIGIKAVHGKIRNQTAHLSVLQLELSMIESMNNLRDLMAYDRKLKNAVTKSMIKILDKHGMILKLKIDENHKNFEIESLIPKEIIHLKDKAIKTNQVSEEYCQLVLALLTTNPGNQLN (SEQ ID NO: 62) c2c2-9 6 CarnobacteriumMRMTKVKINGSPVSMNRSKLNGHLVWNGTTNTVNILTKKEQSFA gallinarumASFLNKTLVKADQVKGYKVLAENIFIIFEQLEKSNSEKPSVYLNNIR DSM 4847RLKEAGLKRFFKSKYHEEIKYTSEKNQSVPTKLNLIPLFFNAVDRIQEDKFDEKNWSYFCKEMSPYLDYKKSYLNRKKEILANSIQQNRGFSMPTAEEPNLLSKRKQLFQQWAMKFQESPLIQQNNFAVEQFNKEFANKINELAAVYNVDELCTAITEKLMNFDKDKSNKTRNFEIKKLWKQHPHNKDKALIKLFNQEGNEALNQFNIELGKYFEHYFPKTGKKESAESYYLNPQTIIKTVGYQLRNAFVQYLLQVGKLHQYNKGVLDSQTLQEIGMYEGFQTKFMDACVFASSSLRNIIQATTNEDILTREKFKKELEKNVELKHDLFFKTEIVEERDENPAKKIAMTPNELDLWAIRGAVQRVRNQIFHQQINKRHEPNQLKVGSFENGDLGNVSYQKTIYQKLFDAEIKDIEIYFAEKIKSSGALEQYSMKDLEKLFSNKELTLSLGGQVVAFAPSYKKLYKQGYFYQNEKTIELEQFTDYDFSNDVFKANYYLIKLIYHYVFLPQFSQANNKLFKDTVHYVIQQNKELNTTEKDKKNNKKIRKYAFEQVKLMKNESPEKYMQYLQREMQEERTIKEAKKTNEEKPNYNFEKLLIQIFIKGFDTFLRNFDLNLNPAEELVGTVKEKAEGLRKRKERIAKILNVDEQIKTGDEEIAFWIFAKLLDARHLSELRNEMIKFKQSSVKKGLIKNGDLIEQMQPILELCILSNDSESMEKESFDKIEVFLEKVELAKNEPYMQEDKLTPVKFRFMKQLEKYQTRNFIENLVIENPEFKVSEKIVLNWHEEKEKIADLVDKRTKLHEEWASKAREIEEYNEKIKKNKSKKLDKPAEFAKFAEYKIICEAIENFNRLDHKVRLTYLKNLHYLMIDLMGRMVGFSVLFERDFVYMGRSYSALKKQSIYLNDYDTFANIRDWEVNENKHLFGTSSSDLTFQETAEFKNLKKPMENQLKALLGVTNHSFEIRNNIAHLHVLRNDGKGEGVSLLSCMNDLRKLMSYDRKLKNAVTKAIIKILDKHGMILKLTNNDHTKPFEIESLKPKKIIHLEKSNHSFPMDQVSQEYCDLVKKMLVFTN (SEQ ID NO: 63) c2c2- 7 PaludibacterMRVSKVKVKDGGKDKMVLVHRKTTGAQLVYSGQPVSNETSNILP 10 propionicigenesEKKRQSFDLSTLNKTIIKFDTAKKQKLNVDQYKIVEKIFKYPKQEL WB4PKQIKAEEILPFLNHKFQEPVKYWKNGKEESFNLTLLIVEAVQAQDKRKLQPYYDWKTWYIQTKSDLLKKSIENNRIDLTENLSKRKKALLAWETEFTASGSIDLTHYHKVYMTDVLCKMLQDVKPLTDDKGKINTNAYHRGLKKALQNHQPAIFGTREVPNEANRADNQLSIYHLEVVKYLEHYFPIKTSKRRNTADDIAHYLKAQTLKTTIEKQLVNAIRANIIQQGKTNHHELKADTTSNDLIRIKTNEAFVLNLTGTCAFAANNIRNMVDNEQTNDILGKGDFIKSLLKDNTNSQLYSFFFGEGLSTNKAEKETQLWGIRGAVQQIRNNVNHYKKDALKTVFNISNFENPTITDPKQQTNYADTIYKARFINELEKIPEAFAQQLKTGGAVSYYTIENLKSLLTTFQFSLCRSTIPFAPGFKKVFNGGINYQNAKQDESFYELMLEQYLRKENFAEESYNARYFMLKLIYNNLFLPGFTTDRKAFADSVGFVQMQNKKQAEKVNPRKKEAYAFEAVRPMTAADSIADYMAYVQSELMQEQNKKEEKVAEETRINFEKFVLQVFIKGFDSFLRAKEFDFVQMPQPQLTATASNQQKADKLNQLEASITADCKLTPQYAKADDATHIAFYVFCKLLDAAHLSNLRNELIKFRESVNEFKFHHLLEIIEICLLSADVVPTDYRDLYSSEADCLARLRPFIEQGADITNWSDLFVQSDKHSPVIHANIELSVKYGTTKLLEQIINKDTQFKTTEANFTAWNTAQKSIEQLIKQREDHHEQWVKAKNADDKEKQERKREKSNFAQKFIEKHGDDYLDICDYINTYNWLDNKMHFVHLNRLHGLTIELLGRMAGFVALFDRDFQFFDEQQIADEFKLHGFVNLHSIDKKLNEVPTKKIKEIYDIRNKIIQINGNKINESVRANLIQFISSKRNYYNNAFLHVSNDEIKEKQMYDIRNHIAHFNYLTKDAADFSLIDLINELRELLHYDRKLKNAVSKAFIDLFDKHGMILKLKLNADHKLKVESLEPKKIYHLGSSAKDKPEYQYCTNQVMMAYCNMCRSLLEMKK (SEQ ID NO: 64) c2c2- 9 ListeriaMLALLHQEVPSQKLHNLKSLNTESLTKLFKPKFQNMISYPPSKGAE 11 weihenstephanensisHVQFCLTDIAVPAIRDLDEIKPDWGIFFEKLKPYTDWAESYIHYKQ FSLTTIQKSIEQNKIQSPDSPRKLVLQKYVTAFLNGEPLGLDLVAKKYK R9-0317LADLAESFKVVDLNEDKSANYKIKACLQQHQRNILDELKEDPELNQYGIEVKKYIQRYFPIKRAPNRSKHARADFLKKELIESTVEQQFKNAVYHYVLEQGKMEAYELTDPKTKDLQDIRSGEAFSFKFINACAFASNNLKMILNPECEKDILGKGDFKKNLPNSTTQSDVVKKMIPFFSDEIQNVNFDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYTDSDMQKLMDKDIAKIPDFIEEKLKSSGIIRFYSHDKLQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYDLGLTTFDILEYGEEDFRARYFLTKLVYYQQFMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSTEDTPNHFEKFISQVFIKGFDSHMRSADLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWTFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVGEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDASPEFKVSKCNITEWERQKETIEETIERRKELHNEWEKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLVYVNELHHLLIEILGRYVGYVAIADRDFQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLFVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKFSRLVIKSLEPKKLRHLGEKKIDNGYIETNQVSEEYCGIVKRLLEI (SEQ ID NO: 65) c2c2- 10 ListeriaceaeMKITKMRVDGRTIVMERTSKEGQLGYEGIDGNKTTEIIFDKKKESF 12 bacteriumYKSILNKTVRKPDEKEKNRRKQAINKAINKEITELMLAVLHQEVPS FSLQKLHNLKSLNTESLTKLFKPKFQNMISYPPSKGAEHVQFCLTDIAV M6-PAIRDLDEIKPDWGIFFEKLKPYTDWAESYIHYKQTTIQKSIEQNKI 0635 =QSPDSPRKLVLQKYVTAFLNGEPLGLDLVAKKYKLADLAESFKLV ListeriaDLNEDKSANYKIKACLOQHQRNILDELKEDPELNQYGIEVKKYIQ newyorkensisRYFPIKRAPNRSKHARADFLKKELIESTVEQQFKNAVYHYVLEQG FSL M6-KMEAYELTDPKTKDLQDIRSGEAFSFKFINACAFASNNLKMILNPE 0635CEKDILGKGNFKKNLPNSTTRSDVVKKMIPFFSDELQNVNFDEAIWAIRGSIQQIRNEVYHCKKHSWKSILKIKGFEFEPNNMKYADSDMQKLMDKDIAKIPEFIEEKLKSSGVVRFYRHDELQSIWEMKQGFSLLTTNAPFVPSFKRVYAKGHDYQTSKNRYYNLDLTTFDILEYGEEDFRARYFLTKLVYYQQFMPWFTADNNAFRDAANFVLRLNKNRQQDAKAFINIREVEEGEMPRDYMGYVQGQIAIHEDSIEDTPNHFEKFISQVFIKGFDRHMRSANLKFIKNPRNQGLEQSEIEEMSFDIKVEPSFLKNKDDYIAFWIFCKMLDARHLSELRNEMIKYDGHLTGEQEIIGLALLGVDSRENDWKQFFSSEREYEKIMKGYVVEELYQREPYRQSDGKTPILFRGVEQARKYGTETVIQRLFDANPEFKVSKCNLAEWERQKETIEETIKRRKELHNEWAKNPKKPQNNAFFKEYKECCDAIDAYNWHKNKTTLAYVNELHHLLIEILGRYVGYVAIADRDFQCMANQYFKHSGITERVEYWGDNRLKSIKKLDTFLKKEGLFVSEKNARNHIAHLNYLSLKSECTLLYLSERLREIFKYDRKLKNAVSKSLIDILDRHGMSVVFANLKENKHRLVIKSLEPKKLRHLGGKKIDGGYIETNQVSEEYCGIVKRLL EM (SEQ ID NO: 66) c2c2-12 Leptotrichia MKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIK 13wadei F0279 NPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITNEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQQIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALE (SEQ ID  NO: 67) c2c2- 15Rhodobacter MQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKAL 14 capsulatusIGQWISGTDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFW SB 1003KLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRTSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID (SEQ ID NO: 68) c2c2- 16 RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKAL 15 capsulatusIGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFW R121KLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLFHDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKESEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIRQVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNHMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITQAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID (SEQ ID NO: 69) c2c2- 17 RhodobacterMQIGKVQGRTISEFGDPAGGLKRKISTDGKNRKELPAHLSSDPKAL 16 capsulatusIGQWISGIDKIYRKPDSRKSDGKAIHSPTPSKMQFDARDDLGEAFW DE442KLVSEAGLAQDSDYDQFKRRLHPYGDKFQPADSGAKLKFEADPPEPQAFHGRWYGAMSKRGNDAKELAAALYEHLHVDEKRIDGQPKRNPKTDKFAPGLVVARALGIESSVLPRGMARLARNWGEEEIQTYFVVDVAASVKEVAKAAVSAAQAFDPPRQVSGRSLSPKVGFALAEHLERVTGSKRCSFDPAAGPSVLALHDEVKKTYKRLCARGKNAARAFPADKTELLALMRHTHENRVRNQMVRMGRVSEYRGQQAGDLAQSHYWTSAGQTEIKFSEIFVRLWVGAFALAGRSMKAWIDPMGKIVNTEKNDRDLTAAVNIROVISNKEMVAEAMARRGIYFGETPELDRLGAEGNEGFVFALLRYLRGCRNQTFHLGARAGFLKEIRKELEKTRWGKAKEAEHVVLTDKTVAAIRAIIDNDAKALGARLLADLSGAFVAHYASKEHFSTLYSEIVKAVKDAPEVSSGLPRLKLLLKRADGVRGYVHGLRDTRKHAFATKLPPPPAPRELDDPATKARYIALLRLYDGPFRAYASGITGTALAGPAARAKEAATALAQSVNVTKAYSDVMEGRSSRLRPPNDGETLREYLSALTGETATEFRVQIGYESDSENARKQAEFIENYRRDMLAFMFEDYIRAKGFDWILKIEPGATAMTRAPVLPEPIDTRGQYEHWQAALYLVMHFVPASDVSNLLHQLRKWEALQGKYELVQDGDATDQADARREALDLVKRFRDVLVLFLKTGEARFEGRAAPFDLKPFRALFANPATFDRLFMATPTTARPAEDDPEGDGASEPELRVARTLRGLRQIARYNFTMAVLSDLFAKHKVRDEEVARLAEIEDETQEKSQIVAAQELRTDLHDKVMKCHPKTISPEERQSYAAAIKTIEEHRFLVGRVYLGDHLRLHRLMMDVIGRLIDYAGAYERDTGTFLINASKQLGAGADWAVTIAGAANTDARTQTRKDLAHFNVLDRADGTPDLTALVNRAREMMAYDRKRKNAVPRSILDMLARLGLTLKWQMKDHLLQDATITOAAIKHLDKVRLTVGGPAAVTEARFSQDYLQMVAAVFNGSVQNPKPRRRDDGDAWHKPPKPATAQSQPDQKPPNKAPSAGSRLPPPQVGEVYEGVVVKVIDTGSLGFLAVEGVAGNIGLHISRLRRIREDAIIVGRRYRFRVEIYVPPKSNTSKLNAADLVRID (SEQ ID NO: 70) c2c2-2MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYKKNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKIIDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYLNVGGDKKKSKNKKMLVEKILNINVDLTVEDLADFVIKELEFWNITKRIEKVKKVNNEFLEKRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFTKIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKINDIKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDLEENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKNYEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKIRNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKIQTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLSNINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYPKERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLNDKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVKNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL (SEQ ID NO: 71) c2c2-3 L wadeiMKVTKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIK (Lw2)NPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFDKLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKQNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFKQLNSANVFNYYEKDVIIKYLKNTKFNFVNKNIPFVPSFTKLYNKIEDLRNTLKFFWSVPKDKEEKDAQIYLLKNIYYGEFLNKFVKNSKVFFKITKEVIKINKQRNQKTGHYKYQKFENIEKTVPVEYLAIIQSREMINNQDKEEKNTYIDFIQOIFLKGFIDYLNKNNLKYIESNNNNDNNDIFSKIKIKKDNKEKYDKILKNYEKHNRNKEIPHEINEFVREIKLGKILKYTENLNMFYLILKLLNHKELTNLKGSLEKYQSANKEETFSDELELINLLNLDNNRVTEDFELEANEIGKFLDFNENKIKDRKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAKYKISLKELKEYSNKKNEIEKNYTMQQNLHRKYARPKKDEKFNDEDYKEYEKAIGNIQKYTHLKNKVEFNELNLLQGLLLKILHRLVGYTSIWERDLRFRLKGEFPENHYIEEIFNFDNSKNVKYKSGQIVEKYINFYKELYKDNVEKRSIYSDKKVKKLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLETMLRKLLSYDRKLKNAIMKSIVDILKEYGFVATFKIGADKKIEIQTLESEKIVHLKNLKKKKLMTDRNSEELCELVKVMFEYKALEKRPAATKKAGQAKKKKGSYPYDVPDYAYPYDVPDYAYPYDVPDYA* (SEQ ID  NO: 72) C2C2-4Listeria MWISIKTLIHHLGVLFFCDYMYNRREKKIIEVKTMRITKVEVDRKK seeligeriVLISRDKNGGKLVYENEMQDNTEQIMHHKKSSFYKSVVNKTICRPEQKQMKKLVFSGLLQENSQEKIKVSDVTKLNISNFLNHRFKKSLYYFPENSPDKSEEYRIEINLSQLLEDSLKKQQGTFICWESFSKDMELYINWAENYISSKTKLIKKSIRNNRIQSTESRSGQLMDRYMKDILNKNKPFDIQSVSEKYQLEKLTSALKATFKEAKKNDKEINYKLKSTLQNHERQIIEELKENSELNQFNIEIRKHLETYFPIKKTNRKVGDIRNLEIGEIQKIVNHRLKNKIVQRILQEGKLASYEIESTVNSNSLQKIKIEEAFALKFINACLFASNNLRNMVYPVCKKDILMIGEFKNSFKEIKHKKFIRQWSQFFSQEITVDDIELASWGLRGAIAPIRNEIIHLKKHSWKKFFNNPTFKVKKSKIINGKTKDVTSEFLYKETLFKDYFYSELDSVPELIINKMESSKILDYYSSDQLNQVFTIPNFELSLLTSAVPFAPSFKRVYLKGFDYQNQDEAQPDYNLKLNIYNEKAFNSEAFQAQYSLFKMVYYQVFLPQFTTNNDLFKSSVDFILTLNKERKGYAKAFQDIRKMNKDEKPSEYMSYIQSQLMLYQKKQEEKEKINHFEKFINQVFIKGFNSFIEKNRLTYICHPTKNTVPENDNIEIPFHTDMDDSNIAFWLMCKLLDAKQLSELRNEMIKFSCSLQSTEEISTFTKAREVIGLALLNGEKGCNDWKELFDDKEAWKKNMSLYVSEELLQSLPYTQEDGQTPVINRSIDLVKKYGTETILEKLFSSSDDYKVSAKDIAKLHEYDVTEKIAQQESLHKQWIEKPGLARDSAWTKKYQNVINDISNYQWAKTKVELTQVRHLHQLTIDLLSRLAGYMSIADRDFQFSSNYILERENSEYRVTSWILLSENKNKNKYNDYELYNLKNASIKVSSKNDPQLKVDLKQLRLTLEYLELFDNRLKEKRNNISHFNYLNGQLGNSILELFDDARDVLSYDRKLKNAVSKSLKEILSSHGMEVTFKPLYQTNHHLKIDKLQPKKIHHLGEKSTVSSNQVSNEYCQLVRTLLTMK (SEQ ID NO: 73)

Fusion constructs of each of the C2c2 orthologues with mCherry andoptionally NLS or NES were made and cloned in a mammalian expressionvector (FIG. 1A). The various C2c2 orthologues were transfected inHEK293T cells and cellular localization was evaluated based on mCherryexpression. Representative localizations of different C2c2 orthologueswhen fused to a C-terminal and N-terminal NES, when fused to aC-terminal and N-terminal NLS, or without NES or NLS fusion are shown inFIGS. 1B, 1C, and 1D, respectively. NES fusions efficiently resulted incytoplasmic localization of the C2c2 protein. NLS fusions efficientlyresulted in nuclear localization of the C2c2 protein. Variably, alsonucleolar localization can be observed with NLS fusions. When C2c2 wasnot fused to an NLS or NES, a variable cytoplasmic/nuclear localizationwas observed.

Example 3: Activity of C2c2 (Cas13a) in Eukaryotic Cells

A luciferase targeting assay was performed as indicated in FIG. 2 withdifferent gRNAs directed against Gluc. C2c2 orthologues Leptotrichiawadei F0279 (Lw2) and Listeria newyorkensis FSL M6-0635 (LbFSL) werefused to an NLS or NES or alternatively were not fused to a localizationsignal. Normalized protein expression of luciferase was determined andcompared to non targeting (NT) gRNA. The results are shown in FIG. 4 .Efficient knockdown was apparent. The spacer sequences used in theexperiments as depicted in FIGS. 4A and 4B are as follows:

FIG. 4A

Guide 1 ATCAGGGCAAACAGAACTTTGACTCCca (SEQ ID  NO: 1) Guide 2AGATCCGTGGTCGCGAAGTTGCTGGCCA (SEQ ID  NO: 2) Guide 3TCGCCTTCGTAGGTGTGGCAGCGTCCTG (SEQ ID  NO: 3) Guide NTtagattgctgactaccaagtaatecat (SEQ ID NO: 4)FIG. 4B

Guide 1 TCGCCTTCGTAGGTGTGGCAGCGTCCTG (SEQ ID  NO: 5) Guide NTtagattgctgttctaccaagtaatccat (SEQ ID  NO: 6)

A targeting assay based on GFP expression was performed as indicated inFIG. 2C with different gRNAs directed against EGFP. C2c2 orthologuesLeptotrichia wadei F0279 (Lw2) and Listeria newyorkensis FSL M6-0635(LbFSL) were fused to an NLS or NES or alternatively were not fused to alocalization signal. Normalized expression of GFP was determined andcompared to non targeting (NT) gRNA. The results are shown in FIG. 5 .Efficient knockdown was apparent. The spacer sequences used in theexperiments as depicted in FIGS. 5A and 5B are as follows:

FIG. 5A

Guide 1 tgaacagctcctcgcccttgctcaccat (SEQ ID  NO: 7) Guide 2tcagcttgccgtaggtggcatcgccctc (SEQ ID  NO: 8) Guide 3gggtagcggctgaagcactgcacgccgt (SEQ ID  NO: 9) Guide 4ggtcttgtagttgccgtcgtccttgaag (SEQ ID  NO: 10) Guide 5tactccagcttgtgccccaggatgttgc (SEQ ID  NO: 11) Guide 6cacgctgccgtcctcgatgttgtggcgg (SEQ ID  NO: 12) Guide 7tctttgctcagggcggactgggtgctca (SEQ ID  NO: 13) Guide 8gacttgtacagctcgtccatgccgagag (SEQ ID  NO: 14) Guide NTtagattgctgttctaccaagtaatccat (SEQ ID  NO: 4)FIG. 5B

Guide 2 tcagcttgccgtaggtggcatcgccctc (SEQ ID  NO: 8) Guide 3gggtagcggctgaagcactgcacgccgt (SEQ ID  NO: 9) Guide 4ggtcttgtagttgccgtcgtccttgaag (SEQ ID  NO: 10) Guide 5tactccagcttgtgccccaggatgttgc (SEQ ID  NO: 11) Guide 6cacgctgccgtcctcgatgttgtggcgg (SEQ ID  NO: 12) Guide 7tctttgctcagggcggactgggtgctca (SEQ ID  NO: 13) Guide 8gacttgtacagctcgtccatgccgagag (SEQ ID  NO: 14) Guide NTtagattgctgttctaccaagtaatccat (SEQ ID  NO: 4)

A targeting assay was performed on different endogenous target genes inHEK293 cells with gRNAs directed against endogenous target genes. C2c2Leptotrichia wadei F0279 (Lw2) was fused to an NES. Normalized proteinexpression of the respective target genes was determined (compared tonon targeting (NT) gRNA). The results are shown in FIG. 7 . Efficientknockdown was apparent. The spacer sequences used in the experiments asdepicted in FIG. 7 are as follows:

FIG. 7

CTNNB1 ctgctgccacagaccgagaggcttaaaa (SEQ ID  NO: 15) PPIBtccttgattacacgatggaatttgctgt (SEQ ID  NO: 16) mAPK14tcaaggtggggtcacaggagaagccaaa (SEQ ID  NO: 17) CXCR4atgataatgcaatagcaggacaggatga (SEQ ID  NO: 18) TINCRgcgtgagccaccgcgcctggccggctgt (SEQ ID  NO: 19) PCAT1ccagctgcagatgctgcagtttttggcg (SEQ ID  NO: 20) CAPN1ctggaaatggaagatgccggcatagcca (SEQ ID  NO: 21) LETMD1gatgacacctcacacggaccacccctag (SEQ ID  NO: 22) MAPK14taatactgctccagatatgggtgggcca (SEQ ID  NO: 23) RB1catgaagaccgagttatagaatactata (SEQ ID  NO: 24) TP53ggtgaaatattctccatccagtggtttc (SEQ ID  NO: 25) KRASaatttctcgaactaatgtatagaaggca (SEQ ID  NO: 26)

Example 4: Translation Upregulation with Catalytically Inactive of C2c2Fused to a Translation Activator/Promoter in Eukaryotic Cells

Catalytically inactive C2c2 orthologues Leptotrichia wadei F0279 (Lw)and Listeria newyorkensis FSL M6-0635 (LbFSL) were generated.

Lw C2c2 was fused to an NES or without localization signal andoptionally EIF4E.

LbFSL was fused to an NLS and optionally EIF4E.

Gluc was targeted (cf. FIG. 2 ).

Relative protein expression was evaluated based on comparison betweentargeting with C2c2 with and without EIF4E.

The results are shown in FIG. 10 .

Efficient translation upregulation was apparent. The spacer sequenceused in the experiment as depicted in FIG. 10 istagattgctgttctaccaagtaatccat, and target has a 3×binding sites for thisspacer.

Example 5: Co-Localization of C2c2 and its Target Beta-Actin UponTreatment with NaASO₂

The localization of C2c2 targeting beta-actin under influence of sodiumarsenite (NaAsO₂) was investigated. A fusion construct of Leptotrichiawadei C2c2 with mCherry and NES was made and cloned in a mammalianexpression vector together with a guide targeting beta-actin or anon-targeting guide. Cellular localization of C2c2 was evaluated basedon mCherry expression, stress granules were labeled with G3BP1-GFP. LwC2c2 targeting beta actin was found to localize to stress granules upontreatment with NaAsO₂ (FIG. 11A). This localization was guide-dependentas only seen with beta-acting targeting and not with non-targetingguides (FIG. 11B, 11C).

Example 6: Alternative crRNA Promoters are Used to Boost KnockdownActivity

In order to further increase the interference effect, the crRNA wasplaced under the control of the U6 promoter.

With the aim to improve efficiency of interference by C2c2, expressionof genes targeted using tRNA-crRNA and U6 driven crRNA and shRNA werecompared. Reliable target gene knockdown was observed with comparableefficiency as shRNA (FIG. 12 , FIG. 14 ). Further experiments wereperformed to determine effect of increasing crRNA transfection amount(FIG. 13 ), increasing protein transfection amount (FIG. 15 ) and effectof DR-spacer-DR-spacer constructs (FIG. 16 ). It was found that C2c2outperformed optimized shRNA for corresponding targets on endogenousgenes.

Example 7: Fusion Constructs with C2c2

As demonstrated in FIG. 18 , dLw2C2c2-EIF4E fusion can upregulatetranslation of three genes; Protein levels as measured by band intensityon western blot.

Example 8: Target Induced Non-Specific rNase Activity

C2c2 target induced non-specific rNase activity is useful to detect RNAspecies in samples. In the presence of an RNA target of interest,guide-dependent C2c2 nuclease activity is accompanied by non-specificRNAse activity against collateral targets. For example, a reporter RNAcomprising a fluorescent moiety and a fluorescence quencher isnon-specifically cleaved by activated C2c2. An RNA substrate is taggedwith a fluorescent reporter molecule (fluor) on one end and a quencheron the other. In the absence of C2c2 rNase activity, the physicalproximity of the quencher dampens fluorescence from the fluor to lowlevels. When C2c2 target specific cleavage is activated, the RNAsubstrate is non-specifically cleaved and the fluor and quencher arespatially separated. This causes the fluor to emit a signal when excitedby light of the appropriate wavelength. A schematic of such an assay isshown in FIG. 24A.

Example 9: Biochemical Characterization of Lw2C2c2 (LwaCas13a)

Methodology is essentially as described in Abudayyeh et al. (2016) “C2c2is a single-component programmable RNA-guided RNA-targeting CRISPReffector”; Science 353(6299), DOI: 10.1126/science.aaf5573; and isincorporated herein by reference.

Because of LwaCas13a's desirable cleavage activity in bacterial cells,Applicants explored further biochemical characterization to betterunderstand its cleavage prior to testing in mammalian cells. In vitrocleavage reactions with both LshCas13a and LwaCas13a demonstratedprogrammable target cleavage with a guide encoding a 28 nt spacer and arequirement for Mg²⁺ (FIG. 3I) as well as confirming the in vivoincrease of efficiency of LwaCas13a over LshCas13a. Incubation of singlestranded RNA target (ssRNA 1) with LwaCas13a and guide showed detectablecleavage within 2 minutes with nearly complete cleavage after 30 minutesof incubation, while LwaCas13a without guide had no observable cleavage(FIG. 37 ), and cleavage was dose-dependent with LwaC2c2-guide complexlevels (FIG. 36 ). Given the appearance of stereotyped cleavageproducts, Applicants hypothesized that LwaCas13a cleavage patterns weretarget-dependent, similar to LshCas13a (Abudayyeh, O. O. et al. C2c2 isa single-component programmable RNA-guided RNA-targeting CRISPReffector. Science 353, aaf5573, doi:10.1126/science.aaf5573 (2016)).Incubation with multiple RNA targets with various in silico-predictedsecondary structures (FIG. 42 ) revealed substantially differentcleavage patterns (FIG. 42 ). To determine if LwaCas13a cleavagedepended on base identity in exposed single stranded regions on thetarget, Applicants incubated LwaCas13a on a target (modified ssRNAtarget 4) with homopolymer substitutions in a loop (FIG. 43 ).Applicants found stronger cleavage for targets with C or U substitutions(FIG. 43 ), showing that LwaCas13a has more substrate flexibility thanLshCas13a, which preferentially cleaves at U residues (Abudayyeh, O. O.et al. C2c2 is a single-component programmable RNA-guided RNA-targetingCRISPR effector. Science 353, aaf5573, doi:10.1126/science.aaf5573(2016)). In addition to target RNAse activity, the Cas13 family has beenreported to process its own corresponding pre-crRNA transcript from L.wadei (FIG. 43C) (East-Seletsky, A. et al. Two district rNase activitiesof CRISPR-C2c2 enable guide-RNA processing and RNA detection, Nature538, 270-273 (2016)). Applicants also explored the guide constraints onLwaCas13a cleavage by truncating either the spacer or the direct repeat(DR) sequences. Applicants found that LwaCas13a retained in vitrocleavage activity with spacer lengths as short as 20 nt (FIG. 41 ), andcould cleave with DR truncations as short as 27 nt (FIG. 40 ), althoughone DR length truncation (32 nt) seemed to eliminate activity, possiblydue to secondary structure perturbation. Although guide lengths lessthan 20 nt no longer display catalytic activity, the LwaCas13-guidecomplex could still retain binding activity, allowing for orthogonalapplications with a single catalytic enzyme (Dahlman, J. E. et al.Orthogonal gene knockout and activation with a catalytically active Cas9nuclease. Nat Biotechnol 33, 1159-1161, doi:10.1038/nbt.3390 (2015)).

The Cas13 family has been found to have a dual RNAse activity forprocessing of full-length CRISPR transcripts (East-Seletsky, A. et al.Two distinct rNase activities of CRISPR-C2c2 enable guide processing andRNA detection. Nature 538, 270-273, doi:10.1038/nature19802 (2016)), ina manner similar to Cpf1 (Fonfara, I., Richter, H., Bratovic, M., LeRhun, A. & Charpentier, E. The CRISPR-associated DNA-cleaving enzymeCpf1 also processes precursor CRISPRRNA. Nature 532, 517-521,doi:10.1038/nature17945 (2016); Zetsche, B. et al. Multiplex geneediting by CRISPR-Cpf1 using a single guide array. Nat Biotechnol 35,31-34, doi:10.1038/nbt.3737 (2017)). Applicants found that LwaCas13acould cleave the corresponding CRISPR spacer transcript from L. wadei(FIG. 3J) and this cleavage was concentration dependent (FIG. 15B).Furthermore, LwaCas13a showed collateral activity (FIG. 24B) on RNAproducts separated by gel electrophoresis (FIG. 24C), confirmingprevious characterization of collateral activity by cleavage of aquenched fluorophores (Gootenberg, J. S. et al. Nucleic acid detectionwith CRISPR-Cas13a/C2c2. Science In press (2017)).

Leptotrichia wadei F0279 (Lwa2) C2c2 was used in in vitro assays toevaluate cleavage kinetics (FIG. 36-37 ), dependence of cleavageactivity on the presence of cations (FIG. 38 ), PFS preference (FIG. 39), effect of direct repeat length (FIG. 40 ), effect of spacer length(FIG. 41 ), effect of target RNA sequence and secondary structure (FIG.42 ) and nucleotide cut preference (FIG. 43 ).

Example 10: LwaCas13a can be Reprogrammed to Knockdown Reporter mRNA

Given LwaCas13a's robust RNA cleavage activity and flexible sequencepreference, Applicants decided to evaluate its ability to cleavetranscripts in mammalian cells. Applicants first cloned mammaliancodon-optimized LwaCas13a into mammalian expression vectors with msfGFPfusions on the C- or N-terminus and either a dual-flanking nuclearexport signal (NES) or nuclear localization sequence (NLS) and evaluatedexpression and localization (FIG. 1D). Applicants found thatmsfGFP-fused LwaCas13a constructs expressed well and localizedeffectively to the cytoplasm or nucleus according to the localizationsequence. To evaluate the in vivo cleavage activity of LwaCas13aApplicants developed a dual luciferase reporter system, which expressesboth Gaussia luciferase (Gluc) and Cypridina luciferase (Cluc) underdifferent promoters on the same vector, allowing one transcript to serveas the Cas13a target and the other to serve as a dosing control (FIG.2A). Applicants then designed guides against Gluc and cloned them into atRNA^(Val)-promoter-expressing guide vector. Applicants transfected theLwaCas13a expression vector, guide vector, and dual-luciferase constructinto HEK293FTs and measured luciferase activity at 48 hours posttransfection. Applicants found that nuclear-localized LwaCas13a-msfGFPresulted in the highest levels of knockdown (75.7% for guide 1, 72.9%for guide 2), comparable to position-matched shRNA controls (78.3% forguide 1, 51.1% for guide 2) (FIG. 6B), which control for accessibilityand sequence in the target region. Because of the superior cleavage ofthe LwaCas13a-msfGFP-NLS construct, Applicants used this design for allfurther knockdown experiments. The nuclear localized LwaCas13a-msfGFPalso fared better than mCherry-fused versions, likely due to theenhanced stability offered by the msfGFP. The ability to manipulateLwaCas13a activity by engineered fusions highlights the flexibility ofthe Cas13a tool. LwaCas13a is also capable of knockdown in the A375melanoma cell line (FIG. 6A, FIG. 9 ), demonstrating the versatility ofCas13. Applicants also found thatLwaCas13a yields the best Glucknockdown with a spacer length of 28 nt (73.8%) (FIG. 6C) and thatknockdown is dose-responsive to both the protein and guide or crRNAtransfected vector amounts (FIG. 56A, 56B). Guide expression is notsensitive to promoter choice, and guide or crRNAs expressed from thetRNA^(Val) or U6 promoters result in similar levels of Gluc knockdown(66.3% for tRNA^(Val), 74.5% for U6) (FIG. 56C).

Applicants next tested knockdown on three endogenous genes: KRAS, CXCR4,and PPIB, and found that varying levels of knockdown, and for 2 of 3genes, LwaCas13a knockdown (40.4% for PPIB, 83.9% for CXCR4, 57.5% forKRAS) was similar to RNAi with position-matched shRNAs (63.0% for PPIB,73.9% for CXCR4, 44.3% for KRAS) (FIG. 30 ). Applicants also found thatendogenous gene knockdown was flexible to guide promoter choice, withsimilar levels of knockdown for guides expressed from the tRNA^(Val) orU6 promoters (86.7% for tRNA^(Val), 77.6% for U6) (FIG. 56D). Applicantsalso found that LwaCas13a is capable of knockdown in the A375 melanomacell line (FIG. 56E). To expand the versatility of LwaCas13a knockdown,Applicants designed guides against transcripts for rice (Oryza sativa)genes EPSPS, HCT, and PDS and co-transfected the LwaCas13a and guidevectors into O. sativa protoplasts (FIG. 3P). After transfection,Applicants observed >50% knockdown of for all three genes and 7 out of 9guides tested, with maximal knockdown of 78.0% (FIG. 3Q). Together,these results suggest that LwaCas13a is able to mediate similar levelsof RNA knockdown as RNAi. Further exploration of additional members ofthe Cas13 family may reveal proteins able to achieve even more potentknockdown effect.

Example 11: LwaCas13a Knockdown Screening of Reporter and EndogenousTranscripts

To comprehensively characterize the dependence of RNA context on theefficiency of LwaCas13a knockdown, Applicants harnessed theprogrammability of LwaCas13a to tile guides along the length of theGluc, KRAS, PPIB or Cluc transcripts (FIG. 32A). The Gluc and Clucscreens revealed guides with greater than 60% knockdown (FIG. 32 ), andthat a majority of Gluc targeting guides had more than 50% knockdownwith up to 83% maximal knockdown. To compare LwaCas13a knockdown withRNAi, Applicants selected the top three performing guides against Glucand Cluc and compared them to position-matched shRNAs. Applicants foundthat five out of six top performing guides achieved significantly higherlevels of knockdown (p<0.05) than their matched shRNA (FIG. 32D).

Having demonstrated robust knockdown on reporter genes, Applicants nextexplored whether Cas13a could be engineered to target endogenoustranscripts via tiling of two genes, KRAS and PPIB. Applicants foundthat, while knockdown efficiency was transcript dependent, Applicantscould still find guides capable of achieving 50% knockdown on eithertarget with maximal knockdown of 85% and 75% for KRAS and PPIB,respectively (FIG. 49A, 49B). Applicants also found that endogenous geneknockdown was flexible to guide expression design, with similar levelsof knockdown for crRNAs expressed from the tRNA^(Val) or U6 promoters(FIG. 56D).

To further understand the efficiency of LwaCas13 knockdown versus RNAi,Applicants compared a variety of guides to shRNA constructs that wereposition matched to the same target region. Applicants selected the topthree guides from each of the endogenous tiling screens (KRAS and PPIB)and observed robust knockdown with Cas13a (53.7%-88.8%) equivalent tolevels attained by shRNA knockdown (61.8%-95.2%), with shRNA better for2 out of 6 guides (p<0.01) and Cas13a better for 2 out of 6 guides(p<0.01) (FIG. 49H).

Example 12: LwaCas13a Knockdown is Optimal at Accessible Sites in theTarget Transcript

Since Applicants found that LshCas13a activity was governed by targetaccessibility in E. coli (Abudayyeh, O. O. et al. C2c2 is asingle-component programmable RNA-guided RNA-targeting CRISPR effector.Science 353, aaf5573, doi:10.1126/science.aaf5573 (2016)), Applicantsdecided to investigate whether LwaCas13a activity was increased forguides located in regions of accessibility along the four transcriptstargeted in our guide tiling screens. Applicants first found that themost effective guides seemed to cluster into defined regions (FIG. 58A)and by comparing the pair-wise distances between effective guides to thenull distribution, Applicants observed guides are significantly morecloser together than would be expected by chance on all four transcripts(FIG. 58A). These initial clustering results suggest that regions ofaccessibility may be enriched for better LwaCas13a cleavage activity.

To confirm that transcript accessibility influenced LwaCas13a activity,Applicants computationally predicted accessibility of all target regionsacross each of the transcripts and found that these computationalpredictions were partially correlated to knockdown efficiency (FIG.58B-D). Across the four targeted transcripts, predicted targetaccessibility could explain some of the variation in targeting efficacy(4.4%-16% of the variation in knockdown), indicating that whileaccessibility is a determinant of knockdown efficiency, other factorssuch as base-identity, sequence properties and protein binding to theRNA may also play important roles in targeting efficacy. More extensivescreening in the future will likely be able to elucidate thesemechanisms more clearly.

Example 13: Comparison of LwaCas13a Knockdown and RNAi on EndogenousTranscripts

To further understand the efficiency of LwCas13 knockdown versus RNAi,Applicants compared a variety of guides to shRNA constructs that werematched to the same target region. Applicants first selected the topthree guides from each of the endogenous tiling screens (KRAS and PPIB)and observed robust knockdown with Cas13a with knockdown equivalent toshRNA for almost every guide (FIG. 49C). To further compare to shRNA,Applicants also designed Cas13a crRNAs in regions of accessibilitypredicted by the RNAxs algorithm for KRAS, PPIB, and CXCR4 and foundcomparable levels of knockdown to shRNA (FIG. 48D).

Example 14: LwaCas13a Knockdown Screening of MALAT1 lncRNA

Because LwaCas13a can be engineered for cellular localization, it hasversatility for which compartments of the cell can be targeted for RNAknockdown. Applicants designed 93 guides tiled evenly across the entirelncRNA MALAT1 transcript, which is nuclear localized, and transfectedthese guides with nuclear-localized LwaCas13a. Applicants found varyinglevels of knockdown, with up to as much as about 40% to 50% knockdown inone experiment (FIG. 49E). Compared against position-matched shRNA,which showed no detectable knockdown (p>0.05), Cas13a achievedsignificantly higher levels of knockdown (39.0-66.5%, p<0.05) (FIG.49J). Applicants also tiled the lncRNA XTST transcript, and found anaverage of 22.0% and a maximum of 83.9% knockdown across all guides(FIG. 56F).

Example 15: Multiplexed Knockdown of Endogenous Transcripts

Other CRISPR effectors with CRISPR array processing activity, such asCpf1, have been leveraged for multiplexed gene editing by expressingmany guides under one promoter (Zetsche, B. et al. Multiplex geneediting by CRISPR-Cpf1 using a single guide array. Nat Biotechnol 35,31-34, doi:10.1038/nbt.3737 (2017)). Because LwaCas13a can process itsown array, Applicants decided to test multiplexed delivery of LwaCas13aguides as a CRISPR array expressed under a single promoter. Applicantsdesigned five different guides against the endogenous PPIB, CXCR4, KRAS,TINCR, and PCAT transcripts, and delivered the targeting system as aCRISPR array with 28 nt guides flanked by 36 nt direct repeats (DR),representing an unprocessed DR and a truncated spacer, under expressionof the U6 promoter. With this approach, Applicants found levels ofknockdown for each gene that were comparable to single or pooled guidecontrols (FIG. 49F).

Because of concerns that off-target LwaCas13a activity might be causingnon-specific knockdown of the five transcripts targeted by the CRISPRarray, Applicants designed an experiment with multiplexed delivery ofthree guides against PPIB, CXCR4, and KRAS and three variants where eachone of the three guides was replaced with a non-targeting guide.Applicants found that in each case where a guide was absent from thearray, there was no significant knockdown of the transcript targeted bythe missing guide and only the targeted transcripts were knocked down byLwaCas13a, demonstrating that knockdown is not due to nonspecificdegradation of the transcripts, but is in fact due to specific,multiplexed knockdown by LwaCas13a (FIG. 49G).

Example 16: LwaCas13a Knockdown is Sensitive to Mismatches

Specificity is a central concern for nucleic acid targeting tools, andthe specificity of both RNAi and Cas9 DNA-targeting systems (Mali, P. etal. CAS9 transcriptional activators for target specificity screening andpaired nickases for cooperative genome engineering. Nat Biotechnol 31,833-838, doi:10.1038/nbt.2675 (2013); Fu, Y. et al. High-frequencyoff-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat Biotechnol 31, 822-826, doi:10.1038/nbt.2623 (2013); Pattanayak, V.et al. High-throughput profiling of off-target DNA cleavage revealsRNA-programmed Cas9 nuclease specificity. Nat Biotechnol 31, 839-843,doi:10.1038/nbt.2673 (2013); Hsu, P. D. et al. DNA targeting specificityof RNA-guided Cas9 nucleases. Nat Biotechnol 31, 827-832,doi:10.1038/nbt.2647 (2013); Doench, J. G. et al. Optimized sgRNA designto maximize activity and minimize off-target effects of CRISPR-Cas9. NatBiotechnol 34, 184-191, doi:10.1038/nbt.3437 (2016)) has beenextensively characterized. The initial characterization of LshCas13ashowed that it could be sensitive to as few as two mismatches in vitro(Abudayyeh, O. O. et al. C2c2 is a single-component programmableRNA-guided RNA-targeting CRISPR effector. Science 353, aaf5573,doi:10.1126/science.aaf5573 (2016)), and specificity profiling ofLwaCas13a via the collateral effect (Gootenberg, J. S. et al. Nucleicacid detection with CRISPR-Cas13a/C2c2. Science In press (2017))revealed that discrimination could be achieved at double-nucleotideresolution, with single-nucleotide resolution in the seed region of theguide:target duplex. To investigate the specificity of Cas13a in vivo,Applicants introduced mismatches into guides targeting either Gluc (FIG.29A) or the endogenous genes CXCR4, KRAS, and PPIB (FIG. 29B, FIG.59A-59B). Applicants found that for all transcripts, the central regionof the guide:target duplex was most sensitive to single mismatches, inagreement with the previous in vitro characterizations of Cas13aspecificity (Abudayyeh, O. O. et al. C2c2 is a single-componentprogrammable RNA-guided RNA-targeting CRISPR effector. Science 353,aaf5573, doi:10.1126/science.aaf5573 (2016)). While knockdown wasreduced in the seed region, Applicants found that single mismatches werenot sufficient for substantial levels of specificity on some targets,such as Gluc. Tiling of consecutive double mismatches for spacersagainst Gluc (FIG. 29A) revealed that double mismatches resulted in upto 8-fold reduction of activity, showing the promise of Cas13a as aspecific in vivo targeting tool. Applicants also investigated the effectof non-consecutive double mismatches and found that most doublemismatches reduced the knockdown from 80.4% to less than 60%, except fordouble mismatches located in either the 5′ or 3′ distal ends of theguide sequence (FIG. 59C).

For further characterization of Cas13a across multiple specificityparameters in vitro, Applicants used detection of collateral activity(Gootenberg, J. S. et al. Nucleic acid detection withCRISPR-Cas13a/C2c2. Science In press (2017)) as a proxy for directCas13a activity. Given results from Cas9 experiments showing thatspecificity could be increased by shorter spacer lengths (Fu, Y.,Sander, J. D., Reyon, D., Cascio, V. M. & Joung, J. K. ImprovingCRISPR-Cas nuclease specificity using truncated guide RNAs. NatBiotechnol 32, 279-284, doi:10.1038/nbt.2808 (2014)), Applicantswondered whether spacer length had an effect on Cas13a specificityagainst two targets that differ by a single mismatch. Applicants foundthat while shorter spacers have reduced activity (FIG. 59D), as expectedfrom our in vivo LwaCas13a results, shorter spacers also had improvedsingle base-mismatch distinction (FIG. 59D,E). Applicants next exploredif specificity could be improved by designing an additional syntheticmismatch in the spacer sequence, as this approach has successfully beenused for single-mismatch distinction in vitro with LwaCas13a(Gootenberg, J. S. et al. Nucleic acid detection withCRISPR-Cas13a/C2c2. Science In press (2017)). Applicants found that,compared to full-length spacers (FIG. 59F, 59G), spacers truncated toeither 23 nt (59H, 59I) or 20 nt (FIG. 59J, 59K) had less overallactivity but substantially increased specificity. Taken together, the invitro and in vivo engineering of LwaCas13a show promise for its use as aspecific knockdown tool. The ability to engineer guides to confersingle-base specificity should facilitate allele-specific transcriptknockdown by LwaCas13a.

Example 17: Transcript Knockdown with LwaCas13a is Highly Specific

To comprehensively understand if there are any off-target effects ofLwaCas13a knockdown, Applicants performed transcriptome-wide mRNAsequencing. Applicants targeted the Gluc transcript with LwaCas13a or aposition matched-shRNA construct, and found significant knockdown of thetarget transcript (FIG. 29D-I). Similar results were found for the samecomparison on two endogenous genes KRAS and PPIB (FIG. 29D-II, 29D-III).shRNA conditions had more transcriptome-wide variation and weakercorrelation between targeting and non-targeting controls than LwaCas13aconditions, suggesting more off-targets in the shRNA targetingexperiment. Applicants further characterized the number of significantoff-targets by differential expression analysis and found hundreds ofoff-targets in each of the shRNA conditions but zero-off targets inLwaCas13a conditions (FIG. 30A), despite comparable levels of knockdownof the target transcripts (30.5%, 43.5%, and 64.7% for shRNA, 62.6%,27.1%, and 29.2% for Cas13a, for Gluc, KRAS, and PPIB, respectively)(FIG. 30B). Applicants performed additional analysis of the Gluctargeting RNA-seq comparisons, and found that the dominant source ofvariability in shRNA conditions was due to differences between targetingand non-targeting conditions in individual replicates (average Kendall'stau=0.917) (FIG. 60C-60E). When individual replicates of the samecondition were compared, there were much higher correlations and lessvariability (average Kendall's tau=0.941), indicating that the variationobserved is from consistent off-target effects of a given shRNAconstruct. When this analysis is applied across all RNA-seq librariesanalyzed for the three genes, all LwaCas13a conditions have highcorrelations with each other despite different guide sequences due tothe narrow spreads of the transcript distributions. In contrast, thesets of three replicates for each of the shRNA conditions have higherintra-set correlation than between shRNA conditions due to the amount ofoff-target variation for each different shRNA sequence (FIG. 61A, 61B).Furthermore, when the distribution of standard deviations for each guidecondition is compared against each shRNA condition across the threetranscripts, there is significantly more variation observed in the shRNAconditions (p<10⁻¹⁹², 2-sided K-S test) (FIG. 61C).

Example 18: LwaCas13a Displays No Observable Collateral Activity inMammalian Cells

The collateral activity of Cas13a has been directly observedbiochemically in vitro and indirectly through growth suppression inbacteria. Because the multiplexed leave-one-out and RNA-seq analysessuggested a lack of non-specific RNA degradation and thus collateralactivity in mammalian cells, Applicants wanted to see if reduction inglobal off-target expression due to collateral activity occurred inknockdown experiments. Applicants analyzed the gene controls in theluciferase and endogenous knockdown experiments to see if there was anyvariation in the controls as a result of target transcript knockdown.From the initial Gluc tiling experiment, it was clear that while manyguides displayed significant knockdown of Gluc, there was littlevariation in Cluc levels (FIG. 62A, 62B). Applicants then decided toanalyze the correlations between on-target knockdown to on-targetexpression or on-target knockdown to off-target expression (theluciferase control or GAPDH in the case of endogenous targeting).Applicants found that for each of the four targets, there wassignificant positive correlation between on-target knockdown andon-target expression (Gluc: R=0.89, p<0.0001; PPIB: R=0.81, p<0.0001;KRAS: R=0.52, p<0.0001) while much weaker or no correlation between theon-target knockdown and control gene expression (Gluc: R=−0.078, p>0.05;PPIB: R=−0.058, p>0.05; KRAS: R=−0.51, p<0.0001) (FIG. 61C-61J),indicating that there was no detectable off-target knockdown.

The lack of any significant correlation between control expression andknockdown suggests that there is little or no collateral activity ofLwaCas13a in mammalian cells. Applicants wanted to further investigatethis by seeing if any growth restriction of cells during transcriptknockdown would be seen as previously observed in bacteria (Abudayyeh,O. O. et al. C2c2 is a single-component programmable RNA-guidedRNA-targeting CRISPR effector. Science 353, aaf5573,doi:10.1126/science.aaf5573 (2016)). Applicants transfected LwaCas13awith multiple guides against Gluc and either with or without selectionfor 72 hours and then measured knockdown immediately before measuringcell viability and LwaCas13a expression via GFP fluorescence. Applicantsobserved significant levels of knockdown for all five Gluc targetingguides (FIG. 30C), but no significant differences in cell growth or GFPfluorescence between the targeting guides and a non-targeting guidecontrol (FIG. 30D, 30E).

The collateral activity of Cas13a has been directly observedbiochemically in vitro and indirectly through growth suppression inbacteria, but the extent of this activity in mammalian cells is unclear.Applicants saw no sequence-specific off-target LwaCas13a activity in ourRNA sequencing experiments, and LwaCas13a-mediated knockdown of targetedtranscripts did not affect the growth of mammalian cells expressingsimilar levels of LwaCas13a (FIG. 29G). Additionally, there were nodetectable gene expression changes, indicating that the presence ofLwaCas13a targeting does not lead to an observable cell stress responseat the transcriptomic level (FIG. 29A, FIG. 60A, 60B) (Subramanian, A.et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles, Proc. Natl. Acad. Sci USA102, 15545-15550 (2005)). In summary, although Applicants cannot ruleout the possibility that low levels of uniform collateral activitycleavage may be occurring, Applicants see no detectable collateralactivity across the four following observations: 1) in all of our tilingexperiments, Applicants observed no significant correlation betweentarget transcript knockdown and the in-line control gene knockdown (FIG.62 ), 2) Applicants see minimal disturbance to the transcriptome in ourRNA sequencing analysis and no significant off-targets (FIG. 29A), 3) inthe leave one-out-multiplexing experiments Applicants do not seeknockdown of the excluded gene (FIG. 49L), and 4) Applicants do not seephenotypic effects on cellular growth or stress due to LwaCas13atargeting (FIG. 29G).

Example 19: dCas13a Programmably Binds Transcripts in Mammalian Cells

As a programmable RNA-binding protein could serve as the foundation fora wide range of applications, Applicants explored whether LwaCas13acould be engineered as a catalytically inactive variant (dCas13a).Previous studies have demonstrated that inactivation of LshCas13a viamutation of catalytic residues eliminated RNAse activity, yet maintainedRNA-binding (Abudayyeh, O. O. et al. C2c2 is a single-componentprogrammable RNA-guided RNA-targeting CRISPR effector. Science 353,aaf5573, doi:10.1126/science.aaf5573 (2016)). Applicants mutatedcatalytic arginine residues in LwaCas13a to generate dCas13a (FIG. 44A)and found that targeting of dCas13a to a 5′ UTR upstream of a reportercoding sequence resulted in reduced translation and reporter geneexpression (FIG. 63A). To quantify RNA binding by dCas13a, Applicantsperformed RNA immunoprecipitation (RIP) (FIG. 58B) using guidescontaining the 36 nt DR and 28 nt spacers and found that pulldown ofdCas13a targeted to either luciferase transcripts (FIG. 44A) or ACTBmRNA (FIG. 63B) resulted in significant enrichment of the correspondingtarget over non-targeting controls (7.8-11.2× enrichment for luciferaseand 2.1 3× enrichment for ACTB; p<0.05), validating dCas13a as areprogrammable RNA binding protein.

Example 20: Negative Feedback Imaging of Transcripts with dCas13a

To engineer dCas13a for in vivo imaging and reduce background noise dueto unbound protein, Applicants incorporated a negative-feedback systembased upon zinc finger self-targeting and KRAB domain repression (Gross,G. G. et al. Recombinant probes for visualizing endogenous synapticproteins in living neurons. Neuron 78, 971-985,doi:10.1016/j.neuron.2013.04.017 (2013)) (FIG. 44B). Fusing a zincfinger, KRAB domain, and NLS to dCas13a resulted in a negative feedbackconstruct (dCas13a-NF). When dCas13a-NF is not bound to its targettranscript, it localizes to nucleus and represses its own expression.Upon transcript binding, dCas13a-NF is exported into the cytoplasm,thereby increasing expression, although it is also possible that newlytranslated dCas13a-NF remains resident in the cytoplasm. In comparisonto dCas13a, which showed modest levels of cytoplasmic translocation (orretention) as a result of transcript binding, dCas13a-NF effectivelytranslocated or re-localized when targeted to ACTB mRNA (FIG. 63E). Tofurther characterize the degree of translocation of dCas13a-NF,Applicants targeted ACTB transcripts with two guides and found that bothguides increased translocation compared to a non-targeting guide(3.1-3.7× cellular/nuclear signal ratio; p<0.0001) (FIG. 44C, FIG.11C-11E). Quantification of translocation showed that targeting guidesresulted in significantly more fluorescence fraction in the cytoplasmthan a non-targeting guide, showing the utility of dCas13a-NF as atranscript imaging tool. To further validate dCas13a-NF imaging,Applicants analyzed the correlation of dCas13a-NF signal to ACTB mRNAfluorescent in situ hybridization (FISH) signal (FIG. 64A) and foundthat there was significant correlation and signal overlap for thetargeting guides versus the non-targeting guide conditions (R=0.27 and0.30 for guide 1 and 2, respectively, and R=0.00 for the non-targetingguide condition; p<0.0001 (FIG. 64B).

Example 21: dCas13a Imaging of Stress Granules in Live Cells

Oxidative stress results in the aggregation of polyadenylatedtranscripts and proteins into stress granules within the cytoplasm(Nelles, D. A. et al. Programmable RNA Tracking in Live Cells withCRISPR/Cas9. Cell 165, 488-496, doi:10.1016/j.cell.2016.02.054 (2016);Unsworth, H., Raguz, S., Edwards, H. J., Higgins, C. F. & Yague, E. mRNAescape from stress granule sequestration is dictated by localization tothe endoplasmic reticulum. FASEBJ24, 3370-3380, doi:10.1096/fj.09-151142(2010)), and the development of stress granules has been associated withmany pathologies, including cancer, neurodegenerative disease, andmyopathies (Wyss-Coray, T. Ageing, neurodegeneration and brainrejuvenation. Nature 539, 180-186, doi:10.1038/nature20411 (2016);Protter, D. S. & Parker, R. Principles and Properties of StressGranules. Trends Cell Biol 26, 668-679, doi:10.1016/j.tcb.2016.05.004(2016)). Applicants investigated the accumulation of mRNA into stressgranules by combining dCas13a-NF imaging of transcripts withvisualization of a well known marker of stress granules, G3BP1(Tourriere, H. et al. The RasGAP-associated endoribonuclease G3BPassembles stress granules. J Cell Biol 160, 823-831,doi:10.1083/jcb.200212128 (2003)) (FIG. 48A). To confirm mRNA trackingin fixed samples, Applicants co-transfected either of two ACTB targetingcrRNAs or guides with dCas13a-NF, induced stress granule formation withsodium arsenite, and visualized G3BP1 with immunofluorescence (FIG.48B). dCas13a-NF translocated in or re-localized to the cytoplasm forthe targeting conditions as expected, and Applicants found significantcorrelations between the dCas13a-NF signal and the G3BP1 fluorescencecompared to the non-targeting control (R=0.49 and 0.50 for guide 1 andguide 2, respectively, and 0.08 for the non-targeting guide; p<0.001)(FIG. 48C, 48D), suggesting the ability of dCas13a-NF to track stressgranule formation. Given co-localization in fixed samples, Applicantsnext performed stress granule tracking in live cells. Applicantstransfected ACTB targeting guide and non-targeting guide withdCas13a-NF, induced stress granule formation with sodium arsenite 24hours post-transfection, and imaged the live cells over time (FIG. 48E).Using G3BP1-RFP fusion as a stress granule marker, Applicants found thatthe dCas13a-NF targeted to ACTB localized to significantly more stressgranules per cell over time than the corresponding non-targeting control(p<0.05) (FIG. 48F).

Example 22: Discussion

The class 2 type VI CRISPR-Cas effector Cas13a can be effectivelyreprogrammed with crRNAs or guides to knockdown or bind transcripts inmammalian cells. Applicants identified LwaCas13a as the most active offifteen Cas13a orthologs for RNA cleavage in bacteria and harnessed itfor mammalian RNA knockdown with levels comparable to RNAi. Applicantsfound that there was no detectable PFS through bacterial screening andthat guide activity was not influenced by PFS. LwaCas13a is sensitive tomismatches in the spacer:target duplex in vivo, and this sensitivitytranslates into high specificity of knockdown compared to RNAi.Applicants also showcase unique attributes of LwaCas13a as an RNAknockdown tool, including the ability to further engineer and optimizethe protein, multiplexed delivery of guides, and knockdown of nuclearlncRNAs. Importantly, Applicants observe no collateral activity ofLwaCas13a, a feature that is highly active in vitro and useful for manyapplications, such as diagnostics (Gootenberg, J. S. et al. Nucleic aciddetection with CRISPR-Cas13a/C2c2. Science In press (2017)).Furthermore, Applicants show that LwaCas13a can be renderedcatalytically inactive, such that it can be used as a programmable RNAbinding platform, and Applicants demonstrate its utility for trackingtranscript accumulation in stress granules in live cells.

Importantly, Applicants observe no collateral activity of LwaCas13a inmammalian cells, a feature that Applicants observed in vitro andharnessed for diagnostics applications (Gootenberg, J. S. et al. Nucleicacid detection with CRISPR-Cas13a/C2c2, Science, in press (2017)).Collateral activity has been hypothesized to be part of a programmedcell death/dormancy pathway in native bacterial cells, which wouldremove infected cells from the population or provide infected cells timeto adapt and overcome infection, supplementing the on-target viraltranscript cleavage activity of Cas13a. The lack of collateral activityin mammalian cells does not preclude the possibility of its existence inthe native cellular context.

There are numerous opportunities for refinement and diversification ofRNA-targeting tools based upon Cas13 family members. In vivocharacterization of additional Cas13 proteins, such as Cas13b (Smargon,A. A. et al. Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNaseDifferentially Regulated by Accessory Proteins Csx27 and Csx28. Mol Cell65, 618-630 e617, doi:10.1016/j.molcel.2016.12.023 (2017)) or Cas13c(Shmakov, S. et al. Diversity and evolution of class 2 CRISPR-Cassystems. Nat Rev Microbiol 15, 169-182, doi:10.1038/nrmicro.2016.184(2017)), may yield further improvements in cleavage or binding capacityand enable applications requiring orthogonal RNA binding proteins,including multi-color imaging. Additionally, smaller orthologs wouldallow for size-constrained delivery options such as Adeno-associatedviral vectors (Ran, F. A. et al. In vivo genome editing usingStaphylococcus aureus Cas9. Nature 520, 186-191,doi:10.1038/nature14299, nature14299 [pii](2015)). Lastly, explorationof the diversity of Cas13 members coupled with increased structural data(Liu, L. et al. Two Distant Catalytic Sites Are Responsible for C2c2RNase Activities. Cell 168, 121-134 e112, doi:10.1016/j.cell.2016.12.031(2017)) may allow for either bioinformatics-(Zinn, E. et al. In SilicoReconstruction of the Viral Evolutionary Lineage Yields a Potent GeneTherapy Vector. Cell Rep 12, 1056-1068, doi:10.1016/j.celrep.2015.07.019(2015)) or structure-(Kleinstiver, B. P. et al. High-fidelityCRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.Nature 529, 490-495, doi:10.1038/nature16526 (2016); Slaymaker, I. M. etal. Rationally engineered Cas9 nucleases with improved specificity.Science 351, 84-88, doi:10.1126/science.aad5227 (2016)) guided rationaldesign. Improved RNA binding tools will allow additionalfunctionalizations, including imaging via reconstitution of splitfluorophores (Ozawa, T., Natori, Y., Sato, M. & Umezawa, Y. Imagingdynamics of endogenous mitochondrial RNA in single living cells. NatMethods 4, 413-419, doi:10.1038/nmeth1030 (2007)), translationalmodulation (De Gregorio, E., Preiss, T. & Hentze, M. W. Translationdriven by an eIF4G core domain in vivo. EMBO J 18, 4865-4874,doi:10.1093/emboj/18.17.4865 (1999); Adamala, K. P., Martin-Alarcon, D.A. & Boyden, E. S. Programmable RNA-binding protein composed of repeatsof a single modular unit. Proc Natl Acad Sci USA 113, E2579-2588,doi:10.1073/pnas.1519368113 (2016); Campbell, Z. T., Valley, C. T. &Wickens, M. A protein-RNA specificity code enables targeted activationof an endogenous human transcript. Nat Struct Mol Biol 21, 732-738,doi:10.1038/nsmb.2847 (2014); Cao, J. et al. Light-inducible activationof target mRNA translation in mammalian cells. Chem Commun (Camb) 49,8338-8340, doi:10.1039/c3cc44866e (2013); Cooke, A., Prigge, A.,Opperman, L. & Wickens, M. Targeted translational regulation using thePUF protein family scaffold. Proc Natl Acad Sci USA 108, 15870-15875,doi:10.1073/pnas.1105151108 (2011)), RNA base editing (Nishikura, K.A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol CellBiol 17, 83-96, doi:10.1038/nrm.2015.4 (2016); Wedekind, J. E., Dance,G. S., Sowden, M. P. & Smith, H. C. Messenger RNA editing in mammals:new members of the APOBEC family seeking roles in the family business.Trends Genet 19, 207-216, doi:10.1016/S0168-9525(03)00054-4 (2003)),epitranscriptomic perturbation (Harcourt, E. M., Kietrys, A. M. & Kool,E. T. Chemical and structural effects of base modifications in messengerRNA. Nature 541, 339-346, doi:10.1038/nature21351 (2017)), targetedinduction of apoptosis based on RNA expression levels (Rider, T. H. etal. Broad-spectrum antiviral therapeutics. PLoS One 6, e22572,doi:10.1371/journal.pone.0022572 (2011)), or splicing modulation (Wang,Y., Cheong, C. G., Hall, T. M. & Wang, Z. Engineering splicing factorswith designed specificities. Nat Methods 6, 825-830,doi:10.1038/nmeth.1379 (2009)).

RNA knockdown with Cas13a can be applied to perturbing RNAs in multiplebiological contexts, including genome-wide pooled knockdown screening,interrogation of lncRNA and nascent transcript function, allele-specificknockdown, and RNA viral therapeutics. In addition, dCas13a andderivatives enable RNA pulldown to study RNA-protein interactions,tracking of transcripts in live cells, and targeted destruction of cellsbased on RNA levels, which would be useful for studying specific cellpopulations or killing cancerous cells. Applicants have shown Cas13 tobe a robust platform for both programmable knockdown and binding of RNAsin mammalian and plant cells, and this platform may be extended to othereukaryotic organisms. CRISPR-Cas13 coupled with creative engineeringapproaches will be a powerful platform for nucleic acid baseddiagnostics and therapeutics and can usher a revolution for studying thetranscriptome.

Example 23: Split Designs for Apoptosis

It is often desirable to deplete or kill cells based on transcriptionalsignatures or specific gene expression, either for basic biologyapplications to study the role of specific cells types or fortherapeutic applications such as cancer or senescent cell clearance(Baker, D. J., Childs, B. G., Durik, M., Wijers, M. E., Sieben, C. J.,Zhong, J., Saltness, R. A., Jeganathan, K. B., Verzosa, G. C., Pezeshki,A., et al. (2016). Naturally occurring p16(Ink4a)-positive cells shortenhealthy lifespan. Nature 530, 184-189.). This targeted cell killing canbe achieved by fusing split apoptotic domains to Cas13 proteins, whichupon binding to the transcript are reconstituted, leading to death ofcells specifically expressing targeted genes or sets of genes. Incertain embodiments, the apoptotic domains may be split Caspase 3(Chelur, D. S., and Chalfie, M. (2007). Targeted cell killing byreconstituted caspases. Proc. Natl. Acad. Sci. U.S.A. 104, 2283-2288.).Other possibilities are the assembly of Caspases, such as bringing twoCaspase 8 (Pajvani, U. B., Trujillo, M. E., Combs, T. P., Iyengar, P.,Jelicks, L., Roth, K. A., Kitsis, R. N., and Scherer, P. E. (2005). Fatapoptosis through targeted activation of caspase 8: a new mouse model ofinducible and reversible lipoatrophy. Nat. Med. 11, 797-803.) or Caspase9 (Straathof, K. C., Pule, M. A., Yotnda, P., Dotti, G., Vanin, E. F.,Brenner, M. K., Heslop, H. E., Spencer, D. M., and Rooney, C. M. (2005).An inducible caspase 9 safety switch for T-cell therapy. Blood 105,4247-4254.) effectors in proximity via Cas13 binding. It is alsopossible to reconstitute a split TEV (Gray, D. C., Mahrus, S., andWells, J. A. (2010). Activation of specific apoptotic caspases with anengineered small-molecule-activated protease. Cell 142, 637-646.) viaCas13 binding on a transcript. This split TEV can be used in a varietyof readouts, including luminescent and fluorescent readouts (Wehr, M.C., Laage, R., Bolz, U., Fischer, T. M., Grunewald, S., Scheek, S.,Bach, A., Nave, K.-A., and Rossner, M. J. (2006). Monitoring regulatedprotein-protein interactions using split TEV. Nat. Methods 3, 985-993.).One embodiment involves the reconstitution of this split TEV to cleavemodified pro-caspase 3 or pro-caspase 7 (Gray, D. C., Mahrus, S., andWells, J. A. (2010). Activation of specific apoptotic caspases with anengineered small-molecule-activated protease. Cell 142, 637-646),resulting in cell death.

Example 24: Split Designs for Imaging

While mentioned in the current application, additional split-fluorophoreconstructs were designed for imaging with reduced background viareconstitution of a split fluorophore upon binding of 2 Cas13 proteinsto a transcript. These split proteins include iSplit (Filonov, G. S.,and Verkhusha, V.V. (2013). A near-infrared BiFC reporter for in vivoimaging of protein-protein interactions. Chem. Biol. 20, 1078-1086.),Split Venus (Wu, B., Chen, J., and Singer, R. H. (2014). Background freeimaging of single mRNAs in live cells using split fluorescent proteins.Sci. Rep. 4, 3615.), and Split superpositive GFP (Blakeley, B. D.,Chapman, A. M., andMcNaughton, B. R. (2012). Split-superpositive GFPreassembly is a fast, efficient, and robust method for detectingprotein-protein interactions in vivo. Mol. Biosyst. 8, 2036-2040.).

Example 25: Split Designs for Additional Transcriptional Activity,Luciferase

Additional possible split fusions that could be constituted by Cas13proteins could include luciferase for luminescent imaging (Kim, S. B.,Ozawa, T., Watanabe, S., and Umezawa, Y. (2004). High-throughput sensingand noninvasive imaging of protein nuclear transport by usingreconstitution of split Renilla luciferase. Proc. Natl. Acad. Sci.U.S.A. 101, 11542-11547.) or split transcription factors to driveexpression of genes of genetic circuits in an RNA-sensing based manner.Possible split transcription factors include split-ubquitin basedsystems, such as the split-ubiquitin-LexA system (Petschnigg, J.,Groisman, B., Kotlyar, M., Taipale, M., Zheng, Y., Kurat, C. F., Sayad,A., Sierra, J R., Mattiazzi Usaj, M., Snider, J., et al. (2014). Themammalian-membrane two-hybrid assay (MaMTH) for probing membrane-proteininteractions in human cells. Nat. Methods 11, 585-592.)

Example 26: Identification of C2c2 Orthologs

The following C2c2 orthologues may be codon optimized for expression inmammalian cells.

C2c2 orthologue Code Multi Letter Leptotrichia buccalis C-1013-b C2-17Lbu Herbinix hemicellulosilytica C2-18 Hhe [Eubacterium] rectale C2-19Ere Eubacteriaceae bacterium CHKCI004 C2-20 Eba Blautia sp.Marseille-P2398 C2-21 BSm Leptotrichia sp. oral taxon 879 str. F0557C2-22 Lsp Lachnospiraceae bacterium NK4A144 C2-23 NK4A144 RNA-bindingprotein S1 Chloroflexus C2-24 aggregans Demequina aurantiaca C2-25Thalassospira sp. TSL5-1 C2-26 SAMN04487830_13920 Pseudobutyrivibrio sp.C2-27 OR37 SAMN02910398_00008 Butyrivibrio sp. C2-28 YAB3001 Blautia sp.Marseille-P2398 C2-29 Leptotrichia sp. Marseille-P3007 C2-30 Bacteroidesihuae C2-31 SAMN05216357_1045 Porphyromonadaceae C2-32 bacteriumKH3CP3RA Listeria riparia C2-33 Insolitispirillum peregrinum C2-34

The protein sequences of the above species are listed in the Tablebelow.

C2-17 Leptotrichia MKVTKVGGISHKKYTSEGRLVKSESEENRTDERLSALLNMRLDMbuccalis C- YIKNPSSTETKENQKRIGKLKKFFSNKMVYLKDNTLSLKNGKKENI 1013-bDREYSETDILESDVRDKKNFAVLKKIYLNENVNSEELEVFRNDIKKKLNKINSLKYSFEKNKANYQKINENNIEKVEGKSKRNIIYDYYRESAKRDAYVSNVKEAFDKLYKEEDIAKLVLEIENLTKLEKYKIREFYHEIIGRKNDKENFAKIIYEEIQNVNNMKELIEKVPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQDGEIATSDFIARNRQNEAFLRNIIGVSSVAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYVSGEVDKIYNENKKNEVKENLKMFYSYDFNMDNKNEIEDFFANIDEAISSIRHGIVHFNLELEGKDIFAFKNIAPSEISKKMFQNEINEKKLKLKIFRQLNSANVFRYLEKYKILNYLKRTRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEIIDAQIYLLKNIYYGEFLNYFMSNNGNFFEISKEIIELNKNDKRNLKTGFYKLQKFEDIQEKIPKEYLANIQSLYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQEFDKFLKKYEQNNNIKIPYEINEFLREIKLGNILKYTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKIADKAGYKISIEELKKYSNKKNEIEKNHKMQENLHRKYARPRKDEKFTDEDYESYKQAIENIEEYTHLKNKVEFNELNLLQGLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFENKKNVKYKGGQIVEKYIKFYKELHQNDEVKINKYSSANIKVLKQEKKDLYIRNYIAHFNYIPHAEISLLEVLENLRKLLSYDRKLKNAVMKSVVDILKEYGFVATFKIGADKKIGIQTLESEKIVHLKNLKKKKLMTDRNSEELCKLVKIMFEYKME EKKSEN (SEQ ID NO: 74)C2-18 Herbinix MKLTRRRISGNSVDQKITAAFYRDMSQGLLYYDSEDNDCTDKVIEhemicellulosilytica SMDFERSWRGRILKNGEDDKNPFYMFVKGLVGSNDKIVCEPIDVDSDPDNLDILINKNLTGFGRNLKAPDSNDTLENLIRKIQAGIPEEEVLPELKKIKEMIQKDIVNRKEQLLKSIKNNRIPFSLEGSKLVPSTKKMKWLFKLIDVPNKTFNEKMLEKYWEIYDYDKLKANITNRLDKTDKKARSISRAVSEELREYHKNLRTNYNRFVSGDRPAAGLDNGGSAKYNPDKEEFLLFLKEVEQYFKKYFPVKSKHSNKSKDKSLVDKYKNYCSYKVVKKEVNRSIINQLVAGLIQQGKLLYYFYYNDTWQEDFLNSYGLSYIQVEEAFKKSVMTSLSWGINRLTSFFIDDSNTVKFDDITTKKAKEAIESNYFNKLRTCSRMQDHFKEKLAFFYPVYVKDKKDRPDDDIENLIVLVKNAIESVSYLRNRTFHFKESSLLELLKELDDKNSGQNKIDYSVAAEFIKRDJENLYDVFREQIRSLGIAEYYKADMISDCFKTCGLEFALYSPKNSLMPAFKNVYKRGANLNKAYIRDKGPKETGDQGQNSYKALEEYRELTWYIEVKNNDQSYNAYKNLLQLIYYHAFLPEVRENEALITDFINRTKEWNRKETEERLNTKNNKKHKNFDENDDITVNTYRYESIPDYQGESLDDYLKVLQRKQMARAKEVNEKEEGNNNYIQFIRDVVVWAFGAYLENKLKNYKNELQPPLSKENIGLNDTLKELFPEEKVKSPFNIKCRFSISTFIDNKGKSTDNTSAEAVKTDGKEDEKDKKNIKRKDLLCFYLFLRLLDENEICKLQHQFIKYRCSLKERRFPGNRTKLEKETELLAELEELMELVRFTMPSIPEISAKAESGYDTMIKKYFKDFIEKKVFKNPKTSNLYYHSDSKTPVTRKYMALLMRSAPLHLYKDIFKGYYLITKKECLEYIKLSNIIKDYQNSLNELHEQLERIKLKSEKQNGKDSLYLDKKDFYKVKEYVENLEQVARYKHLQHKINFESLYRIFRIHVDIAARMVGYTQDWERDMHFLFKALVYNGVLEERRFEAIFNNNNDDNNDGRIVKKIQNNLNNKNRELVSMLCWNKKLNKNEFGAIIWKRNPIAHLNHFTQTEQNSKSSLESLINSLRILLAYDRKRQNAVTKTINDLLLNDYHIRIKWEGRVDEGQIYFNIKEKEDTENEPIIHLKHLHKKDCYIYKNSYMFDKQKEWICNGIKEEVYDKSILKCIGNLFKFDYEDKN KSSANPKHT (SEQ ID NO: 75)C2-19 [Eubacterium] MLRRDKEVKKLYNVFNQIQVGTKPKKWNNDEKLSPEENERRAQQ rectaleKNIKMKNYKWREACSKYVESSQRIINDVIFYSYRKAKNKLRYMRKNEDILKKMQEAEKLSKFSGGKLEDFVAYTLRKSLVVSKYDTQEFDSLAAMVVFLECIGKNNISDHEREIVCKLLELIRKDFSKLDPNVKGSQGANIVRSVRNQNMIVQPQGDRFLFPQVYAKENETVTNKNVEKEGLNEFLLNYANLDDEKRAESLRKLRRILDVYFSAPNHYEKDMDITLSDNIEKEKFNVWEKHECGKKETGLFVDIPDVLMEAEAENIKLDAVVEKRERKVLNDRVRKQNIICYRYTRAVVEKYNSNEPLFFENNAINQYWIHHIENAVERILKNCKAGKLFKLRKGYLAEKVWKDAINLISIKYIALGKAVYNFALDDIWKDKKNKELGIVDERIRNGITSFDYEMIKAHENLQRELAVDIAFSVNNLARAVCDMSNLGNKESDFLLWKRNDIADKLKNKDDMASVSAVLQFFGGKSSWDINIFKDAYKGKKKYNYEVRFIDDLRKAIYCARNENFHFKTALVNDEKWNTELFGKIFERETEFCLNVEKDRFYSNNLYMFYQVSELRNMLDHLYSRSVSRAAQVPSYNSVIVRTAFPEYITNVLGYQKPSYDADTLGKWYSACYYLLKEIYYNSFLQSDRALQLFEKSVKTLSWDDKKQQRAVDNFKDHFSDIKSACTSLAQVCQIYMTEYNQQNNQIKKVRSSNDSIFDQPVYQHYKVLLKKAIANAFADYLKNNKDLFGFIGKPFKANEIREIDKEQFLPDWTSRKYEALCIEVSGSQELQKWYIVGKFLNARSLNLMVGSMRSYIQYVTDIKRRAASIGNELHVSVHDVEKVEKVVVQVIEVCSLLASRTSNQFEDYFNDKDDYARYLKSYVDFSNVDMPSEYSALVDFSNEEQSDLYVDPKNPKVNRNIVHSKLFAADHILRDIVEPVSKDNIEEFYSQKAEIAYCKIKGKEITAEEQKAVLKYQKLKNRVELRDIVEYGEIINELLGQLINWSFMRERDLLYFQLGFHYDCLRNDSKKPEGYKNIKVDENSIKDAILYQIIGMYVNGVTVYAPEKDGDKLKEQCVKGGVGVKVSAFHRYSKYLGLNEKTLYNAGLEIFEVVAEHEDIINLRNGIDHFKYYLGDYRSMLSIYSEVFDRFFTYDIKYQKNVLNLLQNILLRHNVIVEPILESGFKTIGEQTKPGAKLSIRSIKSDTFQYKVKGGTLITDAKDERYLETIRKILYYAENEEDNLKKSVVVTNADKYEKNKESDDQNKQKEKKNKDNKGKKNEETKSDAEKNNNERLSYNPFANLNFKLSN (SEQ ID NO: 76) C2-20 EubacteriaceaeMKISKESHKRTAVAVMEDRVGGVVYVPGGSGIDLSNNLKKRSMD bacteriumTKSLYNVFNQIQAGTAPSEYEWKDYLSEAENKKREAQKMIQKAN CHKCI004YELRRECEDYAKKANLAVSRIIFSKKPKKIFSDDDIISHMKKQRLSKFKGRMEDFVLIALRKSLVVSTYNQEVFDSRKAATVFLKNIGKKNISADDERQIKQLMALIREDYDKWNPDKDSSDKKESSGTKVIRSIEHQNMVIQPEKNKLSLSKISNVGKKTKTKQKEKAGLDAFLKEYAQIDENSRMEYLKKLRRLLDTYFAAPSSYIKGAAVSLPEMNFSSELNVWERHEAAKKVNINFVEIPESLLNAEQNNNKINKVEQEHSLEQLRTDIRRRNITCYHFANALAADERYHTLFFENMAMNQFWIHHMENAVERILKKCNVGTLFKLRIGYLSEKVWKDMLNLLSIKYIALGKAVYHFALDDIWKADIWKDASDKNSGKINDLTLKGISSFDYEMVKAQEDLQREMAVGVAFSTNNLARVTCKMDDLSDAESDFLLWNKEAIRRHVKYTEKGEILSAILQFFGGRSLWDESLFEKAYSDSNYELKFLDDLKRAIYAARNETFHFKTAAIDGGSWNTRLFGSLFEKEAGLCLNVEKNKFYSNNLVLFYKQEDLRWLDKLYGKECSRAAQIPSYNTILPRKSFSDFMKQLLGLKEPVYGSAILDQWYSACYYLFKEVYYNLFLQDSSAKALFEKAVKALKGADKKQEKAVESFRKRYWEISKNASLAEICQSYITEYNQQNNKERKVRSANDGMFNEPIYQHYKMLLKEALKMAFASYIKNDKELKFVYKPTEKLFEVSQDNFLPNWNSEKYNTLISEVKNSPDLQKWYIVGKFMNARMLNLLLGSMRSYLQYVSDIQKRAAGLGENQLHLSAENVGQVKKWIQVLEVCLLLSVRISDKFTDYFKDEEEYASYLKEYVDFEDSAMPSDYSALLAFSNEGKIDLYVDASNPKVNRNIIQAKLYAPDMVLKKVVKKISQDECKEFNEKKEQIMQFKNKGDEVSWEEQQKILEYQKLKNRVELRDLSEYGELINELLGQLINWSYLRERDLLYFQLGFHYSCLMNESKKPDAYKTIRRGTVSIENAVLYQIIAMYINGFPVYAPEKGELKPQCKTGSAGQKIRAFCQWASMVEKKKYELYNAGLELFEVVKEHDNIIDLRNKIDHFKYYQGNDSILALYGEIFDRFFTYDMKYRNNVLNHLQNILLRHNVIIKPIISKDKKEVGRGKMKDRAAFLLEEVSSDRFTYKVKEGERKIDAKNRLYLETVRDILYFPNRAVNDKGEDVIICSKKAQDLNEKKADRDKNHDKSKDTNQKKEGKNQEEKSENKEPYSDRMTWKPFAGIKLE (SEQ ID NO: 77) C2-21 Blautia sp.MKISKVDHVKSGIDQKLSSQRGMLYKQPQKKYEGKQLEEHVRNL Marseille-SRKAKALYQVFPVSGNSKMEKELQIINSFIKNILLRLDSGKTSEEIV P2398GYINTYSVASQISGDHIQELVDQHLKESLRKYTCVGDKRIYVPDIIVALLKSKFNSETLQYDNSELKILIDFIREDYLKEKQIKQIVHSIENNSTPLRIAEINGQKRLIPANVDNPKKSYIFEFLKEYAQSDPKGQESLLQHMRYLILLYLYGPDKITDDYCEEIEAWNFGSIVMDNEQLFSEEASMLIQDRIYVNQQIEEGRQSKDTAKVKKNKSKYRMLGDKIEHSINESVVKHYQEACKAVEEKDIPWIKYISDHVMSVYSSKNRVDLDKLSLPYLAKNTWNTWISFIAMKYVDMGKGVYHFAMSDVDKVGKQDNLIIGQIDPKFSDGISSFDYERIKAEDDLHRSMSGYIAFAVNNFARAICSDEFRKKNRKEDVLTVGLDEIPLYDNVKRKLLQYFGGASNWDDSIIDIIDDKDLVACIKENLYYARNVNFHFAGSEKVQKKQDDILEEIVRKETRDIGKHYRKVFYSNNVAVFYCDEDIIKLMNHLYQREKPYQAQIPSYNKVISKTYLPDLIFMLLKGKNRTKISDPSIMNMFRGTFYFLLKEIYYNDFLQASNLKEMFCEGLKNNVKNKKSEKPYQNFMRRFEELENMGMDFGEICQQIMTDYEQQNKQKKKTATAVMSEKDKKIRTLDNDTQKYKHFRTLLYIGLREAFIIYLKDEKNKEWYEFLREPVKREQPEEKEFVNKWKLNQYSDCSELILKDSLAAAWYVVAHFINQAQLNHLIGDIKNYIQFISDIDRRAKSTGNPVSESTEIQIERYRKILRVLEFAKFFCGQITNVLTDYYQDENDFSTHVGHYVKFEKKNMEPAHALQAFSNSLYACGKEKKKAGFYYDGMNPIVNRNITLASMYGNKKLLENAMNPVTEQDIRKYYSLMAELDSVLKNGAVCKSEDEQKNLRHFQNLKNRIELVDVLTLSELVNDLVAQLIGWVYlRERDMMYLQLGLHYIKLYFTDSVAEDSYLRTLDLEEGSIADGAVLYQIASLYSFNLPMYVKPNKSSVYCKKHVNSVATKFDIFEKEYCNGDETVIENGLRLFENINLHKDMVKFRDYLAHFKYFAKLDESILELYSKAYDFFFSYNIKLKKSVSYVLTNVLLSYFINAKLSFSTYKSSGNKWQHRTTKISVVAQTDYFTYKLRSIVKNKNGVESIENDDRRCEVVNIAARDKEFVDEVCNVINYNSDK (SEQ ID NO: 78) C2-22LeptQtrichia MGNLFGHKRWYEVRDKXDFKIKRKVKVKRNYDGNKYILNINENN sp. oralNKEKIDNNKFIGEFVNYKKNNNVLKEFKRKFHAGNILFKLKGKEEI taxon 879IRIENNDDFLETEEVVLYIEVYGKSEKLKALEITKKKIIDEAIRQGIT str. F0557KDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNINMSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDNKIDVILTNFMEIREKIKSNLEIMGFVKFYLNVSGDKKKSENKKMFVEKILNTNVDLTVEDIVDFIVKELKFWNITKRIEKVKKFNNEFLENRRNRTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKINELIKKLEKELKKGNCDTEIFGIFKKHYKVNFDSKKFSNKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEIEKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIVKMTVNTDDFSRLHAKEELDLELITFFASTNMELNKIFNGKEKVTDFFGFNLNGQKITLKEKVPSFKLNILKKLNFINNENNIDEKLSHFYSFQKEGYLLRNKILHNSYGNIQETKNLKGEYENVEKLIKELKVSDEEISKSLSLDVIFEGKVDIINKINSLKIGEYKDKKYLPSFSKIVLEITRKFREINKDKLFDIESEKIILNAVKYVNKILYEKITSNEENEFLKTLPDKLVKKSNNKKENKNLLSIEEYYKNAQVSSSKGDKKAIKKYQNKVTNAYLEYLENTFTEIIDFSKFNLNYDEIKTKIEERXDNKSKIIIDSISTNINITNDIEYIISIFALLNSNTYINKIRNRFFATSVWLEKQNGTKEYDYENIISILDEVLLINLLRENNITDILDLKNAIIDAKIVENDETYIKNYIFESNEEKLKKRLFCEELVDKEDIRKIFEDENFKFKSFIKKNEIGNFKFVFGILSNLECNSEVEAKKIIGKNSKKLESFIQNIIDEYKSNIRTLFSSEFLEKYKEEIDNLVEDTESENKNKFEKIYYPKEHKNELYIYKKNLFLNIGNPNFDKIYGLISKDIKNVDTKILFDDDIKKNKISEIDAILKNLNDKLNGYSNDYKAKYVNKLKENDDFFAKNIQNENYSSFGEFEKDYNKVSEYKKIRDLVEFNYLNKIESYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYYKFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNLLSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFRLIGNNDILERLMKPKKVSVLELESYNSDYIKNLIIELLTKIENTNDTL (SEQ ID NO: 79) C2-23 LachnospiraceaeMKISKVDHTRMAVAKGNQHRRDEISGILYKDPTKTGSIDFDERFK bacteriumKLNCSAKILYHVFNGIAEGSNKYKNIVDKVNNNLDRVLFTGKSYD NK4A144RKSIIDIDTVLRNVEKINAFDRISTEEREQIIDDLLEIQLRKGLRKGKAGLREVLLIGAGVIVRTDKKQEIADFLEILDEDFNKTNQAKNIKLSIENQGLWSPVSRGEERIFDVSGAQKGKSSKKAQEKEALSAFLLDYADLDKNVRFEYLRKIRRLINLYFYVKNDDVMSLTEIPAEVNLEKDFDIWRDHEQRKEENGDFVGCPDILLADRDVKKSNSKQVKIAERQLRESIREKNIKRYRFSIKTIEKDDGTYFFANKQISVFWIHRIENAVERILGSINDKKLYRLRLGYLGEKVWKDILNFLSIKYIAVGKAVFNFAMDDLQEKDRDIEPGKISENAVNGLTSFDYEQIKADEMLQREVAVNVAFAANNLARVTVDIPQNGEKEDILLWNKSDIKKYKKNSKKGILKSILQFFGGASTWTNMKMFEIAYHDQPGDYEENYLYDIIQIIYSLRNKSFHFKTYDHGDKNWNRELIGKMIEHDAERVISVEREKFHSNNLPMFYKDADLKKILDLLYSDYAGRASQVPAFNTVLVRKNFPEFLRKI)MGYKVHFNNPEVENQWHSAVYYLYKElYYNLFLRDKEVKNLFYTSLKNIRSEVSDKKQKLASDDFASRCEEIEDRSLPEICQIIMTEYNAQNFGNRKWSQRVTEKNKDIFRHYKMLLIKTLAGAFSLYLKQERFAFIGKATPIPYETTDVKNFLPEWKSGMYASFVEEIKNNLDLQEWYIVGRFLNGRMLNQLAGSLRSYIQYAEDIEREAAENRNKLFSKPDEKIEACKKAVRVLDLCIKISTRISAEFTDYFDSEDDYADYLEKYLKYQDDAIKELSGSSYAALDHFCNKDDLKFDIYVNAGQKPILQRNIVMAKLFGPDNILSEVMEKVTESAIREYYDYLKKVSGYRVRGKCSTEKEQEDLLKFQRLKNAVEFRDVTEYAEVINELLGQLISWSYLRERDLLYFQLGFHFYMCLKNKSFKPAEYVDIRRNNGTIIFINAILYQIVSNIYINGLDFYSCDKEGKTLKPIETGKGVGSKIGQFIKYSQYLYNDPSYKLEIYNAGLEVFENIDEHDNITDLRKYVDHFKYYAYGNKMSLLDLYSEFFDRFFTYDMKYQKNVVNVLENILLRHFVIFYPKFGSGKKDVGIRDCKKERAQIEISEQSLTSEDFMFKLDDKAGEEAKKFPARDERYLQTIAKLLYYPNEIEDNINRFMKKGETINKKVQFNRKKKITRKQKNNSSNEVLSSTMGYLFKNIKL (SEQ ID NO: 80) C2-24 ChlorofiexusMTDQVRREEVAAGELADTPLAAAQTPAADAAVAATPAPAEAVAP aggregansTPEQAVDQPATTGESEAPVTTAQAAAHEAEPAEATGASFTPVSEQQPQKPRRLKDLQPGMELEGKVTSIALYGIFVDVGVGRDGLVHISEMSDRRIDTPSELVQIGDTVKVWVKSVDLDARRISLTMLNPSRGEKPRRSRQSQPAQPQPRRQEVDREKLASLKVGEIVEGVITGFAPFGAFADIGVGKDGLIHISELSEGRVEKPEDAVKVGERYQFKVLEIDGEGTRISLSLRRAQRTQRMQQLEPGQIIEGTVSGIATFGAFVDIGVGRDGLVHISALAPHRVAKVEDVVKVGDKVKVKVLGVDPQSKRISLTMRLEEEQPATTAGDEAAEPAEEVTPTRRGNLERFAAAAQTARERSERGERSERGERRERRERRPAQSSPDTYIVGEDDDESFEGNATIEDLLTKFGGSSSRRDRDRRRRHEDDDDEEMERPSNRRQREAIRRTLQQIGYDE (SEQ ID NO: 81) C2-25Demequina MDLTWHALLILFWALLAGFLDTLAGGGGLLTVPALLLTGIPPLQA aurantiacaLGTNKLQSSFGTGMATYQVIRKKRVHWRDVRWPMVWAFLGSAAGAVAVQFIDTDALLIIIPVVLALVAAYFLFVPKSHLPPPEPRMSDPAYEATLVPIIGAYDGAFGPGTGSLYALSGVALRAKTLVQSTALAKTLNFATNFAALIATAFAGHMLWTVGAVMIAGQLIGAYAGSHMLFRVNPLVLRVLIVVMSLGMLIRVLLD (SEQ ID NO: 82) C2-26 ThalassospiraMRIIKPYGRSHVEGVATQEPRRKLRLNSSPDISRDIPGFAQSHDALII sp. TSL5-1AQWISAIDKIATKPKPDKKPTQAQINLRTTLGDAAWQHVMAENLLPAATDPAIREKLHLTWQSKIAPWGTARPQAEKDGKPTPKGGWYERFCGVLSPEAITQNVARQIAKDIYDHLHVAAKRKGREPAKQGESSNKPGKFKPDRKRGLIEERAESIAKNALRPGSHAPCPWGPDDQATYEQAGDVAGQLYAAARDCLEEKKRRSGNRNTSSVQYLPRDLAAKILYAQYGRVFGPDTTIKAALDEQPSLFALHKAIKDCYHRLINDARKRDILRILPRNMAALFRLVRAQYDNRDINALIRLGKVIHYHASEQGKSEHHGTRDYWPSQQDIQNSRFWGSDGQADIKRHEAFSRIWRHIIALASRTLHDWADPHSQKFSGENDDILLLAKDAIEDDVFKAGHYERKCDVLFGAQASLFCGAEDFEKAILKQAITGTGNLRNATFHFKGKVRFEKELQELTKDVPVEVQSAIAALWQKDAEGRTRQIAETLQAVLAGHFLTEEQNRHIFAALTAAMAQPGDVPLPRLRRVLARHDSICQRGRILPLSPCPDRAKLEESPALTCQYTVLKMLYDGPFRAWLAQQNSTILNHYIDSTIARTDKAARDMNGRKLAQAEKDLITSRAADLPRLSVDEKMGDFLARLTAATATEMRVQRGYQSDGENAQKQAAFIGQFECDVIGRAFADFLNQSGFDFVLKLKADTPQPDAAQCDVTALIAPDDISVSPPQAWQQVLYFILHLVPVDDASHLLHQIRKWQVLEGKEKPAQIAHDVQSVLMLYLDMHDAKFTGGAALHGIEKFAEFFAHAADFRAVFPPQSLQDQDRSIPRRGLREIVRFGHLPLLQHMSGTVQITHDNVVAWQAARTAGATGMSPIARRQKQREELHALAVERTARFRNADLQNYMHALVDVIKHRQLSAQVTLSDQVRLHRLMMGVLGRLVDYAGLWERDLYFVVLALLYHHGATPDDVFKGQGKKNLADGQVVAALKPKNRKAAAPVGVFDDLDHYGIYQDDRQSIRNGLSHFNMLRGGKAPDLSHWVNQTRSLVAHDRKLKNAVAKSVIEMLAREGFDLDWGIQTDRGQHILSHGKIRTRQAQHFQKSRLHIVKKSAKPDKNDTVKIRENLHGDAMVERVVQLFAAQVQKRYDRTVEKRLDHLFLKPQDQKGKNGIHTHNGWSKTEKKRRPSRENRKGNHEN (SEQ ID NO: 83) C2-27 SAMN04487830_13920MKFSKESHRKTAVGVTESNGIIGLLYKDPLNEKEKIEDVVNQRANS [pseudobutyrivibrioTKRLFNLFGTEATSKDISRASKDLAKVVNKAIGNLKGNKKFNKKE sp.QITKGLNTKIIVEELKNVLKDEKKLIVNKDIIDEACSRLLKTSFRTA OR37]KTKQAVKMILTAVLIENTNLSKEDEAFVHEYFVKKLVNEYNKTSVKKQIPVALSNQNMVIQPNSVNGTLEISETKKSKETKTTEKDAFRAFLRDYATLDENRRHKMRLCLRNLVNLYFYGETSVSKDDFDEWRDHEDKKQNDELFVKKIVSIKTDRKGNVKEVLDVDATIDAIRTNNIACYRRALAYANENPDVFFSDTMLNKFWIHHVENEVERIYGHINNNTGDYKYQLGYLSEKVWKGIINYLSIKYIAEGKAVYNYAMNALAKDNNSNAFGKLDEKFVNGITSFEYERIKAEETLQRECAVNTAFAANHLANATVDLNEKDSDFLLLKHEDNKDTLGAVARPNILRNILQFFGGKSRWNDFDFSGIDEIQLLDDLLRKMIYSLRNSSFHFKTENIDNDSWNTKLIGDMFAYDFNMAGNVQKDKMYSNNVPMFYSTSDIEKMLDRLYAEVHERASQVPSFNSVFVRKNFPDYLKNDLKITSAFGVDDALKWQSAVYYVCKEIYYNDFLQNPETFTMLKDYVQCLPIDIDKSMDQKLKSERNAHKNFKEAFATYCKECDSLSAICQMIMTEYNNQNKGNRKVISARTKDGDKLIYKHYKMILFEALKNVFTITLEKNINTYGFLKKPKLINNVPAIEEFLPNYNGRQYETLVNRTTEETELQKWYIVGRLLNPKQVNQLIGNFRSYVQYVNDVARRAKQTGNNLSNDNIAWDVKNIIQIFDVCTKLNGVTSNILEDYFDDGDDYARYLKNFVDYTNKNNDHSATLLGDFCAKEIDGIKIGIYHDGTNPIVNRNIIQCKLYGATGIISDLTKDGSILSVDYEIIKKYMQMQKEIKVYQQKGICKTKEEQQNLKKYQELKNIVELRNIIDYSEILDELQGQLINWGYLRERDLMYFQLGFHYLCLHNESKKPVGYNNAGDISGAVLYQIVAMYTNGLSLIDANGKSKKNAKASAGAKVGSFCSYSKEIRGVDKDTKEDDDPIYLAGVELFENINEHQQCINLRNYIEHFHYYAKHDRSMLDLYSEVFDRFFTYDMKYTKNVPNMMYNTLLQHLVVPAFEFGSSEKRLDDNDEQTKPRAMFTLREKNGLSSEQFTYRLGDGNSTVKLSARGDDYLRAVASLLYYPDRAPEGLIRDAEAEDKFAKINHSNPKSDNRNNRGNFKNPKVQWYNNKTKRK (SEQ ID NO: 84) C2-28SAMN02910398_00008 [ButyrivibrioMKISKVDHRKTAVKITDNKGAEGFIYQDPTRDSSTMEQIISNRARS sp.SKVLFNIFGDTKKSKDLNKYTESLIIYVNKAIKSLKGDKRNNKYEEI YAB3001]TESLKTERVLNALIQAGNEFTCSENNIEDALNKYLKKSFRVGNTKSALKKLLMAAYCGYKLSIEEKEEIQNYFVDKLVKEYNKDTVLKYTAKSLKHQNMVVQPDTDNHVFLPSRIAGATQNKMSEKEALTEFLKAYAVLDEEKRHNLRIILRKLVNLYFYESPDFIYPENNEWKEFIDDRKNKTETFVSPVKVNEEKNGKTFVKIDVPATKDLTRLKNIECYRRSVAETAGNPITYFTDFHNISKFWIHHIENEVEKIFALLKSNWKDYQFSVGYISEKVWKEIINYLSIKYIAIGKAVYNYALEDIKKNDGTLNFGVIDPSFYDGINSFEYEKIKAEETFQREVAWVSFAVNHLSSATVKLSEAQSDMLVLNKNDIEKIAYGNTKRNILQFFGGQSKWKEFDFDRYINPVNYTDIDFLFDIKKMVYSLRNESFHFTTTDTESDWNKNLISAMFEYECRRISTVQKNKFFSNNLPLFYGENSLERVLHKLYDDYVDRMSQVPSFGNVFVRKKFPDYMKEIGIKHNLSSEDNLKLQGALYFLYKEIYYNAFISSEKAMKIFVDLVNKLDTNARDDKGRITHEAMAHKNFKDAISHYMTHDCSLADICQKIMTEYNQQNTGHRKKQTTYSSEKNPEIFRHYKMILFMLLQKAMTEYISSEEIFDFIMKPNSPKTDIKEEEFLPQYKSCAYDNLIKLIADNVELQKWYITARLLSPREVNQLIGSFRSYKQFVSDIERRAKETNNSLSKSGMTVDVENITKVLDLCTKLNGRFSNELTDYFDSKDDYAVYVSKFLDFGFKIDEKFPAALLGEFCNKEENGKKIGIYHNGTEPILNSNIIKSKLYGITDVVSRAVKPVSEKLIREYLQQEVKIKPYLENGVCKNKEEQAALRKYQELKNRIEFRDIVEYSEIINELMGQLINFSYLRERDLMYFQLGFHYLCLNNYGAKPEGYYSIVNDKRTIKGAILYQIVAMYTYGLPIYHYVDGTISDRRKNKKTVLDTLNSSETVGAKIKYFIYYSDELFNDSLILYNAGLELFENINEHENIVNLRKYIDHFKYYVSQDRSLLDIYSEVFDRYFTYDRKYKKNVMNLFSNIMLKHFIITDFEFSTGEKTIGEKNTAKKECAKVRIKRGGLSSDKFTYKFKDAKPIELSAKNTEFLDGVARILYYPENVVLTDLVRNSEVEDEKRIEKYDRNHNSSPTRKDKTYKQDVKKNYNKKTSKAFDSSKLDTKSVGNNLSDNPVLKQFLSESKKKR (SEQ ID NO: 85) C2-29 Blautia sp.MKISKVDHVKSGIDQKLSSQRGMLYKQPQKKYEGKQLEEHVRNL Marseille-SRKAKALYQVFPVSGNSKMEKELQIINSFIKNILLRLDSGKTSEEIV P2398GYINTYSVASQISGDHIQELVDQHLKESLRKYTCVGDKRIYVPDIIVALLKSKFNSETLQYDNSELKILIDFIREDYLKEKQIKQIVHSIENNSTPLRIAEINGQKRLIPANVDNPKKSYIFEFLKEYAQSDPKGQESLLQHMRYLILLYLYGPDKITDDYCEEIEAWNFGSIVMDNEQLFSEEASMLIQDRIYVNQQIEEGRQSKDTAKVKKNKSKYRMLGDKIEHSINESVVKHYQEACKAVEEKDIPWIKYISDHVMSVYSSKNRVDLDKLSLPYLAKNTWNTWISFIAMKYVDMGKGVYHFAMSDVDKVGKQDNLIIGQIDPKFSDGISSFDYERIKAEDDLHRSMSGYIAFAVNNFARAICSDEFRKKNRKEDVLTVGLDEIPLYDNVKRKLLQYFGGASNWDDSIIDIIDDKDLVACIKENLYVARNVNFHFAGSEKVQKKQDDILEEIVRKETRDIGKHYRKVFYSNNVAVFYCDEDIIKLMNHLYQREKPYQAQIPSYNKVISKTYLPDLIFMLLKGKNRTKISDPSIMNMFRGTFYFLLKEIYYNDFLQASNLKEMFCEGLKNNVKNKKSEKPYQNFMRRFEELENMGMDFGEICQQIMTDYEQQNKQKKKTATAVMSEKDKKIRTLDNDTQKYKHFRTLLYIGLREAFIIYLKDEKNKEWYEFLREPVKREQPEEKEFVNKWKLNQYSDCSELILKDSLAAAWYVVAHFINQAQLNHLIGDIKNYIQFISDIDRRAKSTGNPVSESTEIQIERYRKILRVLEFAKFFCGQITNVLTDYYQDENDFSTHVGHYVKFEKKNMEPAHALQAFSNSLYACGKEKKKAGFYYDGMNPIVNRNITLASMYGNKKLLENAMNPVTEQDIRKYYSLMAELDSVLKNGAVCKSEDEQKNLRHFQNLKNRIELVDVLTLSELVNDLVAQLIGWVYIRERDMMYLQLGLHYIKLYFTDSVAEDSYLRTLDLEEGSIADGAVLYQIASLYSFNLPMYVKPNKSSVYCKKHVNSVATKFDIFEKEYCNGDETVIENGLRLFENINLHKDMVKFRDYLAHFKYFAKLDESILELYSKAYDFFFSYNIKLKKSVSYVLTNVLLSYFINAKLSFSTYKSSGNKTVQHRTTKISVVAQTDYFTYKLRSIVKNKNGVESIENDDRRCEVVNIAARDKEFVDEVCNVINYNSDK (SEQ ID NO: 86) C2-30Leptotrichia MKITKIDGISHKKYIKEGKLVKSTSEENKTDERLSELLTIRLDTYIK sp.NPDNASEEENRIRRENLKEFFSNKVLYLKDGILYLKDRREKNQLQN Marseille-KNYSEEDISEYDLKNKNNFLVLKKILLNEDINSEELEIFRNDFEKKL P3007DKINSLKYSLEENKANYQKINENNIKKVEGKSKRNIFYNYYKDSAKRNDYINNIQEAFDKLYKKEDIENLFFLIENSKKHEKYKIRECYHKIIGRKNDKENFATIIYEEIQNVNNMKELIEKVPNVSELKKSQVFYKYYLNKEKLNDENIKYVFCHFVEIEMSKLLKNYVYKKPSNISNDKVKRIFEYQSLKKLIENKLLNKLDTYVRNCGKYSFYLQDGEIATSDFIVGNRQNEAFLRNIIGVSSTAYFSLRNILETENENDITGRMRGKTVKNNKGEEKYISGEIDKLYDNNKQNEVKKNLKMFYSYDFNMNSKKEIEDFFSNIDEAISSIRHGIVHFNLELEGKDIFTFKNIVPSQISKKMFHDEINEKKLKLKIFKQLNSANVFRYLEKYKILNYLNRTRFEFVNKNIPFVPSFTKLYSRIDDLKNSLGIYWKTPKTNDDNKTKEITDAQIYLLKNIYYGEFLNYFMSNNGNFFEITKEIIELNKNDKRNLKTGFYKLQKFENLQEKTPKEYLANIQSLYMINAGNQDEEEKDTYIDFIQKIFLKGFMTYLANNGRLSLIYIGSDEETNTSLAEKKQEFDKFLKKYEQNNNIEIPYEINEFVREIKLGKILKYTERLNMFYLILKLLNHKELTNLKGSLEKYQSANKEEAFSDQLELINLLNLDNNRVTEDFELEADEIGKFLDFNGNKVKDNKELKKFDTNKIYFDGENIIKHRAFYNIKKYGMLNLLEKISDEAKYKISIEELKNYSKKKNEIEENHTTQENLHRKYARPRKDEKFTDEDYKKYEKAIRNIQQYTHLKNKVEFNELNLLQSLLLRILHRLVGYTSIWERDLRFRLKGEFPENQYIEEIFNFDNSKNVKYKNGQIVEKYINFYKELYKDDTEKISIYSDKKVKELKKEKKDLYIRNYIAHFNYIPNAEISLLEMLENLRKLLSYDRKLKNAIMKSIVDILKEYGFVVTFKIEKDKKIRIESLKSEEVVHLKKLKLKDNDKKKEPIKTYRNSKELCKLVKVMFEYKMKEKKSEN (SEQ ID NO: 87) C2-31 BacteroidesihuaeMRITKVKVKESSDQKDKMVLIHRKVGEGTLVLDENLADLTAPIIDKYKDKSFELSLLKQTLVSEKEMNIPKCDKCTAKERCLSCKQREKRLKEVRGAIEKTIGAVIAGRDIIPRLNIFNEDEICWLIKPKLRNEFTFKDVNKQVVKLNLPKVLVEYSKKNDPTLFLAYQQWIAAYLKNKKGHIKKSILNNRVVIDYSDESKLSKRKQALELWGEEYETNQRIALESYHTSYNIGELVTLLPNPEEYVSDKGEIRPAFHYKLKNVLQMHQSTVFGTNEILCINPIFNENRANIQLSAYNLEVVKYFEHYFPIKKKKKNLSLNQAIYYLKVETLKERLSLQLENALRMNLLQKGKIKKHEFDKNTCSNTLSQIKRDEFFVLNLVEMCAFAANNIRNIVDKEQVNEILSKKDLCNSLSKNTIDKELCTKFYGADFSQIPVAIWAMRGSVQQIRNEIVHYKAEAIDKIFALKTFEYDDMEKDYSDTPFKQYLELSIEKIDSFFIEQLSSNDVLNYYCTEDVNKLLNKCKLSLRRTSIPFAPGFKTIYELGCHLQDSSNTYRIGHYLMLIGGRVANSTVTKASKAYPAYRFMLKLIYNHLFLNKFLDNHNKRFFMKAVAFVLKDNRENARNKFQYAFKEIRMMNNDESIASYMSYIHSLSVQEQEKKGDKNDKVRYNTEKFIEKVFVKGFDDFLSWLGVEFILSPNQEERDKTVTREEYENLMIKDRVEHSINSNQESHIAFFTFCKLLDANHLSDLRNEWIKFRSSGDKEGFSYNFAIDIIELCLLTVDRVEQRRDGYKEQTELKEYLSFFIKGNESENTVWKGFYFQQDNYTPVLYSPIELIRKYGTLELLKLIIVDEDKITQGEFEEWQTLKKVVEDKVTRRNELHQEWEDMKNKSSFSQEKCSIYQKLCRDIDRYNWLDNKLHLVHLRKLHNLVIQILSRMARFIALWDRDFVLLDASRANDDYKLLSFFNFRDFINAKKTKTDDELLAEFGSKIEKKNAPFIKAEDVPLMVECIEAKRSFYQKVFFRNNLQVLADRNFIAHYNYISKTAKCSLFEMIIKLRTLMYYDRKLRNAVVKSIANVFDQNGMVLQLSLDDSHELKVDKVISKRIVHLKNNNIMTDQVPEEYYKICRRLLEMKK (SEQ ID NO: 88) C2-32SAMN05216357_1045 MEFRDSIFKSLLQKEIEKAPLCFAEKLISGGVFSYYPSERLKEFVGN[Porphyromonadaceae HPFSLFRKTMPFSPGFKRVMKSGGNYQNANRDGRFYDLDIGVYLPbacterium KDGFGDEEWNARYFLMKLIYNQLFLPYFADAENHLFRECVDFVK KH3CP3RA]RVNRDYNCKNNNSEEQAFIDIRSMREDESIADYLAFIQSNIIIEENKKKETNKEGQINFNKFLLQVFVKGFDSFLKDRTELNFLQLPELQGDGTRGDDLESLDKLGAVVAVDLKLDATGIDADLNENISFYTFCKLLDSNHLSRLRNEIIKYQSANSDFSHNEDFDYDRIISIIELCMLSADHVSTNDNESIFPNNDKDFSGIRPYLSTDAKVETFEDLYVHSDAKTPITNATMVLNWKYGTDKLFERLMISDQDFLVTEKDYFVWKELKKDIEEKIKLREELHSLWVNTPKGKKGAKKKNGRETTGEFSEENKKEYLEVCREIDRYVNLDNKLHFVHLKRMHSLLIELLGRFVGFTYLFERDYQYYHLEIRSRRNKDAGVVDKLEYNKIKDQNKYDKDDFFACTFLYEKANKVRNFIAHFNYLTMWNSPQEEEHNSNLSGAKNSSGRQNLKCSLTELINELREVMSYDRKLKNAVTKAVIDLFDKHGMVIKFRIVNNNNNDNKNKHHLELDDIVPKKIMHLRGIKLKRQDGKPIPIQTDSVDPLYCRMWKKLLDLKPTPF (SEQ ID NO: 89) C2-33 ListeriaMHDAWAENPKKPQSDAFLKEYKACCEAIDTYNWHKNKATLVYV ripariaNELHHLLIDILGRLVGYVAIADRDFQCMANQYLKSSGHTERVDSWINTIRKNRPDYIEKLDIFMNKAGLFVSEKNGRNYIAHLNYLSPKHKYSLLYLFEKLREMLKYDRKLKNAVTKSLIDLLDKHGMCVVFANLKNNKHRLVIASLKPKKIETFKWKKIK (SEQ ID NO: 90) C2-34 InsolitispirillumMRIIRPYGSSTVASPSPQDAQPLRSLQRQNGTFDVAEFSRRHPELVL peregrinumAQWVAMLDKIIRKPAPGKNSTALPRPTAEQRRLRQQVGAALWAEMQRHTPVPPELKAVWDSKVHPYSKDNAPATAKTPSHRGRWYDRFGDPETSAATVAEGVRRHLLDSAQPFRANGGQPKGKGVIEHRALTIQNGTLLHHHQSEKAGPLPEDWSTYRADELVSTIGKDARWIKVAASLYQHYGRIFGPTTPISEAQTRPEFVLHTAVKAYYRRLFKERKLPAERLERLLPRTGEALRHAVTVQHGNRSLADAVRIGKILHYGWLQNGEPDPWPDDAALYSSRYWGSDGQTDIKHSEAVSRVWRRALTAAQRTLTSWLYPAGTDAGDILLIGQKPDSIDRNRLPLLYGDSTRHWTRSPGDVWLFLKQTLENLRNSSFHFKTLSAFTSHLDGTCESEPAEQQAAQALWQDDRQQDHQQVFLSLRALDATTYLPTGPLHRIVNAVQSTDATLPLPRFRRVVTRAANTRLKGFPVEPVNRRTMEDDPLLRCRYGVLKLLYERGFRAWLETRPSIASCLDQSLKRSTKAAQTINGKNSPQGVEILSRATKLLQAEGGGGHGIHDLFDRLYAATAREMRVQVGYHHDAEAARQQAEFIEDLKCEVVARAFCAYLKTLGIQGDTFRRQPEPLPTWPDLPDLPSSTIGTAQAALYSVLHLMPVEDVGSLLHQLRRWLVALQARGGEDGTAITATIPLLELYLNRHDAKFSGGGAGTGLRWDDWQVFFDCQATFDRVFPPGPALDSHRLPLRGLREVLRFGRVNDLAALIGQDKITAAEVDRWHTAEQTIAAQQQRREALHEQLSRKKGTDAEVDEYRALVTAIADHRHLTAHVTLSNVVRLHRLMTTVLGRLVDYGGLWERDLTFVTLYEAHRLGGLRNLLSESRVNKFLDGQTPAALSKKNNAEENGMISKVLGDKARRQIRNDFAHFNMLQQGKKTINLTDEINNARKLMAHDRKLKNAITRSVTTLLQQDGLDIVWTMDASHRLTDAKIDSRNAIHLHKTHNRANIREPLHGKSYCRWVAALFGATSTPSATKKSD KIR (SEQ ID NO: 91)

Example 27: Identification of C2c2 Orthologs

Certain Cas13b orthologs are surprisingly similar to C2c2. FIG. 54provides a tree alignment of C2c2 and Cas13b proteins. The followingCas13b proteins may be codon optimized for expression in mammaliancells.

Bergeyella  1 MENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELGKRLK zoohelcumGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEVPIKERKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKSTVLTVKKKKVKTDKTKEILKKSIEKQLDILCQKKLEYLRDTARKIEEKRRNQRERGEKELVAPFKYSDKRDDLIAAIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGDLKIPISKNGVVFLLSLFLTKQEIHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKRSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKIVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDNLKRKYPEAPARKEVLYYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGLVQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKKEKEALIK (SEQ ID NO: 92) Prevotella  2MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKILEEGEI intermediaNRDGYETTLKNTWNEIKDINKKDRLSKLIIKHFPFLEAATYRLNPTDTTKQKEEKQAEAQSLESLRKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEGLLEKMYNIFNASIRLVKEDYQYNKDINPDEDFKHLDRTEEEFNYYFTKDNEGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRENKKKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDREKFRVPIEIADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDTFANGEIKSIDELEEYCKGKDIEIGHLPKQMIAILKDEHKVMATEAERKQEEMLVDVQKSLESLDNQINEEIENVERKNSSLKSGKIASWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLKDTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLKLKEPKTKPKTLVQGWKNGFNLPRGIFTEPIRKWFMKHRENITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYHFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFKSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKNIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTPSKAESKSNTISKLRVEYELGEYQKARIEIIKDMLALEKTLIDKYNSLDTDNFNKMLTDWLELKGEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIETKE (SEQ ID NO: 93) Prevotella  3MQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKHFWAAF buccaeLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDKKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 94) Porphyromonas  4MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFAVFFKPDDFVLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLDEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEGFYKCYLEKKREWLAKIFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 95) Bacteroides  5MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENHIRKWLGD pyogenesVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMFDSDKKSYENRRETAECLDTIDRQKISSLLKEVYGKLQDIRNAFSHYIDDQSVKHTALIISSEMHRFIENAYSFALQKTRARFTGVFYETDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMIASLSKRVRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLQQITKGITDEVRFSLYAPRYAIYNNKIGFVRTSGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPLPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKRVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK (SEQ ID NO: 96) Alistipes sp.  6MSNEIGAFREHQFAYAPGNEKQEEATTATYFNLALSNVEGMMFGEVE ZOR0009SNPDKIEKSLDTLPPAILRQIASFIWLSKEDHPDKAYSTEEVKVIVTDLVRRLCFYRNYFSHCFYLDTQYFYSDELVDTTAIGEKLPYNFHHFITNRLFRYSLPEITXFRWNEGERKYEILRDGLIFFCCLFLKRGQAERFLNELRFFKRTDEEGRIKRTIFTKYCTRESHKHIGIEEQDFLIFQDIIGDLNRVPKVCDGVVDLSKENERYIKNRETSNESDENKARYRLLIREKDKFPYYLMRYIVDFGVLPCITFKQNDYSTKEGRGQFHYQDAAVAQEERCYNFVVRNGNVYYSYMPQAQNVVRISELQGTISVEELRNMVYASINGKDVNKSVEQYLYHLHLLYEKILTISGQTIKEGRVDVEDYRPLLDKLLLRPASNGEELRRELRKLLPKRVCDLLSNRFDCSEGVSAVEKRLKAILLRHEQLLLSQNPALHIDKIKSVIDYLYLFFSDDEKFRQQPTEKAHRGLKDEEFQMYHYLVGDYDSHPLALWKELEASGRLKPEMRKLTSATSLHGLYMLCLKGTVEWCRKQLMSIGKGTAKVEAIADRVGLKLYDKLKEYTPEQLEREVKLVVMHGYAAAATPKPKAQAAIPSKLTELRFYSFLGKREMSFAAFIRQDKKAQKLWLRNFYWENIKTLQKRQAAADAACKKLYNLVGEVERVHTNDKVLVLVAQRYRERLLNVGSKCAVTLDNPERQQKLADVYEVQNAWLSIRFDDLDFTLTHVNLSNLRKAYNLIPRKMLAFKEYLDNRVKQKLCEECRNVRRKEDLCTCCSPRYSNLTSWLKENHSESSIEREAATMMLLDVERKLLSFLLDERRKAIIEYGKFIPFSALVKECRLADAGLCGIRNDVLHDNVISYADAIGKLSAYFPKEASEAVEYIRRTKEVREQRREELMANSSQ (SEQ ID NO: 97)Prevotella  7a MSKECKKQRQEKKRRLQKANFSISLTGKHVFGAYFNMARTNFVKTINsp. MA2016 YILPIAGVRGNYSENQINKMLHALFLIQAGRNEELTTEQKQWEKKLRLNPEQQTKPQKLLFKHFPVLGPMMADVADHKAYLNKKKSTVQTEDETFAMLKGVSLADCLDIICLMADTLTECRNFYTHKDPYNKPSQLADQYLHQEMIAKKLDKVVVASRRILKDREGLSVNEVEFLTGIDHLHQEVLKDEFGNAKVKDGKVMKTFVEYDDFYFKISGKRLVNGYTVTTKDDKPVNVNTMLPALSDFGLLYFCVLFLSKPYAKLFIDEVRLFEYSPFDDKENMIMSEMLSIYRIRTPRLHKIDSHDSKATLAMDIFGELRRCPMELYNLLDKNAGQPFFHDEVKHPNSHTPDVSKRLRYDDRFPTLALRYIDETELFKRIRFQLQLGSFRYKFYDKENCIDGRVRVRRIQKEINGYGRMQEVADKRMDKWGDLIQKREERSVKLEHEELYINLDQFLEDTADSTPYVTDRRPAYNIHANRIGLYWEDSQNPKQYKVFDENGMYIPELVVTEDKKAPIKMPAPRCALSVYDLPAMLFYEYLREQQDNEFPSAEQVIIEYEDDYRKFFKAVAEGKLKPFKRPKEFRDFLKKEYPKLRMADIPKKLQLFLCSHGLCYNNKPETVYERLDRLTLQHLEERELHIQNRLEHYQKDRDMIGNKDNQYGKKSFSDVRHGALARYLAQSMMEWQPTKLKDKEKGHDKLTGLNYNVLTAYLATYGHPQVPEEGFTPRTLEQVLINAHLIGGSNPHPFINKVLALGNRNIEELYLHYLEEELKHIRSRIQSLSSNPSDKALSALPFIHHDRMRYHERTSEEMMALAARYTTIQLPDGLFTPYILEILQKHYTENSDLQNALSQDVPVKLNPTCNAAYLITLFYQTVLKDNAQPFYLSDKTYTRNKDGEKAESFSFKRAYELFSVLNNNKKDTFPFEMIPLFLTSDEIQERLSAKLLDGDGNPVPEVGEKGKPATDSQGNTIWKRRIYSEVDDYAEKLTDRDMKISFKGEWEKLPRWKQDKIIKRRDETRRQMRDELLQRMPRYIRDIKDNERTLRRYKTQDMVLFLLAEKMFTNIISEQSSEFNWKQMRLSKVCNEAFLRQTLTFRVPVTVGETTIYVEQENMSLKNYGEFYRFLTDDRLMSLLNNIVETLKPNENGDLVIRHTDLMSELAAYDQYRSTIFMLIQSIENLIITNNAVLDDPDADGFWVREDLPKRNNFASLLELINQLNNVELTDDERKLLVAIRNAFSHNSYNIDFSLKDVKHLPEVAKGILQHLQSMLGVEITK (SEQ ID NO: 98) Prevotella  7bMSKECKKQRQEKKRRLQKANFSISLTGKHVFGAYFNMARTNFVKTIN sp. MA2016YILPIAGVRGNYSENQINKMLFIALFLIQAGRNEELTTEQKQWEKKLRLNPEQQTKFQKLLFKHFPVLGPMMADVADHKAYLNKKKSTVQTEDETFAMLKGVSLADCLDIICLMADTLTECRNFYTHKDPYNKPSQLADQYLHQEMIAKKLDKVVVASRRILKDREGLSVNEVEFLTGIDFILHQEVLKDEFGNAKVKDGKVMKTFVEYDDFYFKISGKRLVNGYTVTTKDDKPVNVNTMLPALSDFGLLYFCVLFLSKPYAKLFIDEVRLFEYSPFDDKENMIMSEMLSIYRIRTPRLHKIDSHDSKATLAMDIFGELRRCPMELYNLLDKNAGQPFFHDEVKHPNSFITPDVSKRLRYDDRFPTLALRYIDETELFKRIRFQLQLGSFRYKFYDKENCIDGRVRVRRIQKEINGYGRMQEVADKRMDKWGDLIQKREERSVKLEHEELYINLDQFLEDTADSTPYVTDRRPAYNIHANRIGLYWEDSQNPKQYKVFDENGMYIPELVVTEDKKAPIKMPAPRCALSWDLPAMLFYEYLREQQDNEFPSAEQVIIEYEDDYRKFFKAVAEGKLKPFKRPKEFRDFLKKEYPKLRMADIPKKLQLFLCSHGLCYNNKPETVYERLDRLTLQFILEERELHIQNRLEFRYQKDRDMIGNKDNQYGKKSFSDVRHGALARYLAQSMMEWQPTKLKDKEKGHDKLTGLNYNVLTAYLATYGHPQVPEEGFTPRTLEQVLINAHLIGGSNPHPFINKVLALGNRNIEELYLHYLEEELKHIRSRIQSLSSNPSDKALSALPF1FWDRMRYHERTSEEMMALAARYTTIQLPDGLFTPYILEILQKHYTENSDLQNALSQDVPVKLNPTCNAAYLITLFYQTVLKDNAQPFYLSDKTYTRNKDGEKAESFSFKRAYELFSVLNNNKKDTFPFEMIPLFLTSDEIQERLSAKLLDGDGNPVPEVGEKGKPATDSQGNTPWKRRIYSEVDDYAEKLTDRDMKISFKGEWEKLPRWKQDKIIKRRDETRRQMRDELLQRMPRYIRDIKDNERTLRRYKTQDMVLFLLAEKMFTNIISEQSSEFNWKQMRLSKVCNEAFLRQTLTFRVPVWGETTIYVEQENMSLKNYGEFYRFLTDDRLMSLLNNIVETLKPNENGDLVIRHTDLMSELAAYDQYRSTIFMLIQSIENLKIITNNAVLDDPDADGFWVREDLPKRNNFASLLELINQLNNVELTDDERKLLVAIRNAFSHNSYNIDFSLIKDVKHLPEVAKGILQHLQSMLGVEITK (SEQ ID NO: 99) Riemerella  8MEKPLLPNVYTLKHKFFWGAFLNIARHNAFITICHINEQLGLKTPSND anatipestiferDKIVDVVCETWNNILNNDHDLLKKSQLTELILKHFPFLTAMCYHPPKKEGKKKGHQKEQQKEKESEAQSQAEALNPSKLIEALEILVNQLHSLRNYYSHKHKKPDAEKDIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHLNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYFSYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSEDLMLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKREEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYHKTPIRTVYIREEHTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHTSLSSLENEFRALLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYKEALHAPIPLFTVAQPTTEEKDGLGIAEALLKVLREYCEIVKSQI (SEQ ID NO: 100)Prevotella  9 MEDDKKTTGSISYELKDKHFWAAFLNLARHNVYITINHINKLLEIREIDaurantiaca NDEKVLDIKTLWQKGNKDLNQKARLRELMTKHFPFLETAIYTKNKEDKKEVKQEKQAEAQSLESLKDCLFLFLDKLQEARNYYSHYKYSEFSKEPEFEEGLLEKMYNIFGNNIQLVINDYQFINKDINPDEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWMQQKLNGFKDNLENKKKMTHEVFCRSRILMPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGDDREKFKVPFDPADEDYNAEQEPFKNTLIRHQDRFPYFVLRYFDYNEIFKNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKAIVKDLDTYETSNKRYISETTPHYHLENQKIGIRFRNGNKEIWPSLKTNDENNEKSKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEKPNNDEINASIVEGFIKREIRNIFKLYDAFANGEINNIDDLEKYCADKGIPKRHLPKQMVAILYDEHKDMVKEAKRKQKEMVKDTKKLLATLEKQTQKEKEDDGRNVKLLKSGEIARWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQMLQRSLALYNNEEKPTRYFRQVNLIESNNPHPFLKWTKWEECNNILTFYYSYLTKKIEFLNKLKPEDWKKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNNSKEYEKVEALDRVGLVTKVIPLFFKEEYFKDKEENFKEDTQKEFNDCVQPFYNFPYNVGNIHKPKEKDFLHREERIELWDKKKDKFKGYKEKIKSKKLTEKDKEEFRSYLEFQSWNKFERELRLVRNQDIVTWLLCKELIDKLKIDELNIEELKKLRLNNIDTDTAAKKEKNNILNRVMPMELPVTVYEIDDSHKIVKDKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTNSEAESKRNPISKLRVEYELGEYQEARIEIIQDMLALEEKLINKYKDLPTNKFSEMLNSWLEGKDEADKARFQNDVDFLIAVRNAFSHNQYPMHNKIEFANIKPFSLYTANNSEEKGLGIANQLKDKTKETTDKIKKIEKPIETKE (SEQ ID NO: 101) Prevotella 10MEDKPFWAAFFNLARHNVYLTVNHINKLLDLEKLYDEGKHKEIFERE saccharolyticaDIFNISDDVMNDANSNGKKRKLDIKKIWDDLDTDLTRKYQLRELILKHFPFIQPAIIGAQTKERTTIDKDKRSTSTSNDSLKQTGEGDINDLLSLSNVKSMFFLLQILEQLRNYYSHVKHSKSATMPNFDEDLLNWMRYIFIDSVNKVKEDYSSNSVIDPNTSFSHLIYKDEQGKIKPCRYPFTSKDGSINAFGLLFFVSLFLEKQDSIWMQKKIPGFKKASENYMKMTNEVFCRNHILLPKIRLETVYDKDWMLLDMLNEVVRCPLSLYKRLTPAAQNKFKVPEKSSDNANRQEDDNPFSRILVRHQNRFPYFVLRFFDLNEVFTTLRFQINLGCYHFAICKKQIGDKKEVHHLIRTIYGFSRLQNFTQNTRPEEWNTLVKTTEPSSGNDGKTVQGVPLPYISYTIPHYQIENEKIGIKIFDGDTAVDTDIWPSVSTEKQLNKPDKYTLTPGFKADVFLSVHELLPMMFYYQLLLCEGMLKTDAGNAVEKVLIDTRNAIFNLYDAFVQEKINTITDLENYLQDKPILIGHLPKQMIDLLKGHQRDMLKAVEQKKAMLIKDTERRLKLLDKQLKQETDVAAKNTGTLLKNGQIADWLVNDMMRFQPVKRDKEGNPINCSKANSTEYQMLQRAFAFYATDSCRLSRYFTQLHLIHSDNSHLFLSRFEYDKQPNLIAFYAAYLKAKLEFLNELQPQNWASDNYFLLLRAPKNDRQKLAEGWKNGFNLPRGLFTEKIKWFNEHKTIVDISDCDIFKNRVGQVARLIPVFFDKKFKDHSQPFYRYDFNVGNVSKPTEANYLSKGKREELFKSYQNKFKNNIPAEKTKEYREYKNFSLWKKFERELRLIKNQDILIWLMCKNLFDEKIKPKKDILEPRIAVSYIKLDSLQTNTSTAGSLNALAKVVPMTLAIHIDSPKPKGKAGNNEKENKEFTVYIKEEGTKLLKWGNFKTLLADRRIKGLFSYIEHDDIDLKQHPLTKRRVDLELDLYQTCRIDIFQQTLGLEAQLLDKYSDLNTDNFYQMLIGWRKKEGIPRNIKEDTDFLKDVRNAFSHNQYPDSKKIAFRRIRKFNPKELILEEEEGLGIATQMYKEVEKVVNRIKRIELFD (SEQ ID NO: 102)HMPREF9712_03108 11 MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKR[Myroides NTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVD odoratimimusKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSDIVI CCUGENKVLDFLNSSFVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAY 10230]KKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFTEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN (SEQ ID NO: 103) Prevotella 12MEDDKKTTDSIRYELKDKHFWAAFLNLARHNVYITVNHINKILEEDEI intermediaNRDGYENTLENSWNEIKDINKKDRLSKLIIKHFPFLEATTYRQNPTDTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEDLQNKMYNIFDVSIQFVKEDYKHNTDINPKKDFKHLDRKRKGKFHYSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKLEGFKCSNKSYQKMTNEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGVNRKKFYVSFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEVFANLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFDKQNRPDEWKAIVKDSDTFKKKEEKEEEKPYISETTPHYHLENKKIGIAFKNHNIWPSTQTELTNNKRKKYNLGTSIKAEAFLSVHELLPMMFYYLLLKTENTKNDNKVGGKKETKKQGKHKIEAIIESKIKDIYALYDAFANGEINSEDELKEYLKGKDIKIVHLPKQMIAILKNEHKDMAEKAEAKQEKMKLATENRLKTLDKQLKGKIQNGKRYNSAPKSGEIASWLVNDMMRFQPVQKDENGESLNNSKANSTEYQLLQRTLAFFGSEHERLAPYFKQTKLIESSNPHPFLNDTEWEKCSNILSFYRSYLKARKNFLESLKPEDWEKNQYFLMLKEPKTNRETLVQGWKNGFNLPRGFFTEPIRKWFMEHWKSIKVDDLKRVGLVAKVTPLFFSEKYKDSVQPFYNYPFNVGDVNKPKEEDFLHREERIELWDKKKDKFKGYKAKKKFKEMTDKEKEEFHRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKIDELNIKELKKLRLKDINTDTAKKEKNNILNRVMPMELPVTVYKVNKGGYIIKNKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVKTPSEAESESNPISKLRVEYELGKYQNARLDIIEDMLALEKXLIDKYNSLDTDNFHNMLTGWLELKGEAKKARFQNDVKLLTAVRNAFSHNQYPMYDENLFGNTERFSLSSSNIIESKGLDIAAKLKEEVSKAAKKIQNEEDNKKEKET (SEQ ID NO: 104) Capnocytophaga 13MKNIQRLGKGNEFSPFKKEDKFYFGGFLNLANNNIEDFFKEIITRFGIVI canimorsusTDENKKPKETFGEKILNEIFKKDISIVDYEKWVNIFADYFPFTKYLSLYLEEMQFKNRVICFRDVMKELLKTVEALRNFYTHYDHEPIKIEDRVFYFLDKVLLDVSLTVKNKYLKTDKTKEFLNQHIGEELKELCKQRKDYLVGKGKRIDKESEIINGIYNNAFKDFICKREKQDDKENHNSVEKILCNKEPQNKKQKSSATVWELCSKSSSKYTEKSFPNRENDKHCLEVPISQKGIVFLLSFFLNKGEIYALTSNIKGFKAKITKEEPVTYDKNSIRYMATHRMFSFLAYKGLKRKIRTSEINYNEDGQASSTYEKETLMLQMLDELNKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYRDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLCDKRTKQICDTTTEREVKKKITVFGIILSELENKKAIFLNEREEIKGWEVFPNPSYDFPKENISVNYKDFPIVGSILDREKQPVSNKKIGIRVKIADELQREIDKAIKEKKLRNPKNRKANQDEKQKERLVNEIVSTNSNEQGEPVVFIGQPTAYLSMNDIHSVLYEFLINKISGEALETKTVEKIETQIKQIIGKDATTKILKPYTNANSNSINREKLLRDLEQEQQILKTLLEEQQQREKDKKDKKSKRKHELYPSEKGKVAVWLANDIKRFMPKAFKEQWRGYHHSLLQKYLAYYEQSKEELKNLLPKEVFKHFPFKLKGYFQQQYLNQFYTDYLKRRLSYVNELLLNIQNFKNDKDALKATEKECFKFFRKQNYIINPINIQIQSILVYPIFLKRGFLDEKPTMIDREKFKENKDTEEADWFMHYKNYKEDNYQKFYAYPLEKVEEKEKFKRNKQINKQKKNDVYTLMMVEYIIQKIFGDKFVEENPLVLKGIFQSKAERQQNNTHAATTQERNLNGILNQPKDIKIQGKITVKGVKLKDIGNFRKYEIDQRVNTFLDYEPRKEWMAYLPNDWKEKEKQGQLPPNNVIDRQISKYETVRSKILLKDVQELEKIISDEIKEEHRHDLKQGKYYNFKYYILNGLLRQLKNENVENYKVFKLNTNPEKVNITQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKREKEALIK (SEQ ID NO: 105)Porphyromonas 14 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKIDHEHNDEVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRMDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPROMIAILSQEHKDMEEKIRKKLQEMMADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGHDEVASYKEVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSYSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADRINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 106)Prevotella 15 MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENN sp. P5-125ENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLNDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIODKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFINDKEDSAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVEFYERYLIERKFYLTGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLDDDFQTFYQWNRNYRYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKQASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDIMEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGVVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID  NO: 107)Flavobacterium 16 MENLNKILDKENEICISKIFNTKGIAAPITEKALDNIKSKQKNDLNKEAbranchiophilum RLHYFSIGHSFKQIDTKKVFDYVLIEELKDEKPLKFITLQKDFFTKEFSIKLQKLINSIRNINNHYVHNFNDINLNKIDSNVFHFLKESFELAIIEKYYKVNKKYPLDNEIVLFLKELFIKDENTALLNYFTNLSKDEAIEYILTFTITENKIWNINNEHNILNIEKGKYLTFEAMLFLITIFLYKNEANHLLPKLYDFKNNKSKQELFTFFSKKFTSQDIDAEEGHLIKFRDMIQYLNHYPTAWNNDLKLESENKNKIMTTKLIDSIIEFELNSNYPSFATDIQFKKEAKAFLFASNKKRNQTSFSNKSYNEEIRHNPHIKQYRDEIASALTPISFNVKEDKFKIFVKKHVLEEYFPNSIGYEKFLEYNDFTEKEKEDFGLKLYSNPKTNKLIERIDNHKLVKSHGRNQDRFMDFSMRFLAENNYFGKDAFFKCYKFYDTQEQDEFLQSNENNDDVKFHKGKVTTYIKYEEHLKNYSYWDCPFVEENNSMSVKISIGSEEKILKIQRNLMIYFLENALYNENVENQGYKLVNNYYRELKKDVEESIASLDLIKSNPDFKSKYKKILPKRLLHNYAPAKQDKAPENAFETLLKKADFREEOYKKLLKKAEHEKNKEDFVKRNKGKQFKLHFIRKACQMMYFKEKYNTLKEGNAAFEKKDPVIEKRKNKEHEFGHHKNLNITREEFNDYCKWMFAFNGNDSYKKYLRDLFSEKHFFDNQEYKNLFESSVNLEAFYAKTKELFKKWIETNKPTNNENRYTLENYKNLILQKQVFINVYHFSKYLIDKNLLNSENNVIQYKSLENVEYLISDFYFQSKLSIDQYKTCGKLFNKLKSNKLEDCLLYEIAYNYIDKKNVHKIDIQKILTSKIILTINDANTPYKISVPFNKLERYTEMIAIKNQNNLKARFLIDLPLYLSKNKIKKGKDSAGYEIIIKNDLEIEDINTINNKIINDSVKFTEVLMELEKYFILKDKCILSKNYIDNSEIPSLKQFSKVWIKENENEIINYRNlACHFHLPLLETFDNLLLNVEQKFIKEELQNVSTINDLSKPQEYLILLFIKFKHNNFYLNLFNKNESKTIKNDKEVKKNRVLQKFINQVILKKK (SEQ ID NO: 108) Myroides 17MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKR odoratimimusNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSDIVIENKVLDFLNSSFVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVTHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHIELQKLFAYFDTSKSDLELILSNMVMVKDYPIELIDLVKKSRTLVDFLNKYLEARLEYIENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYKQHKEKFADWFYHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCDGLVHIDNVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLLPIGMDVREMLILSTDVKFKKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYAN (SEQ ID NO: 109) Flavobacterium 18MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKEFKTR columnareINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLIPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLERFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLSLVTEYKKNVYLKETSNIDLSRFPLFPNPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNKKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLIKTINTDSSVTFENQPIDIPRLKNALQKELTLTQEKLLNVKEHEIEVDNYNRNKNTYKFKNQPKNKVDDKKLORKYVFYRNEIRQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKGLKNLFLKHGNFIDFYKEYLKLKEDFLSTESTFLENGFIGLPPKILKKELSKRLKYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDIVERDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKAEREKIKADAKAYQKLNDSSLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDARTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKELLKQVQELEKKILDKFYDFSNNASHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILLKYYDYNTEELKEKIKNMDEDEKAKIINTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLANRFVPKKEEEYFATYFNRVFETITKELWENKEKKDKTQV (SEQ ID NO: 110) Porphyromonas 19MTEQNEKPYNGTYYTLEDKHFWAAFLNLARHNAYITLAHIDRQLAY gingivalisSKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELSKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVVADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLEARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERASKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDETLFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMVERIIQA (SEQ ID NO: 111) Porphyromonas 20MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS sp.KADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYG COT-052KKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKL OH4946KDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIGILSQERKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEDRAEEWERGKERFRDLEAWSHSAARRIKDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 112)Prevotella 21 MEDDKKTKESTNMLDNKHFWAAFLNLARHNVYITVNHINKVLELKN intermediaKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLETAIYTKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQEARNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEEFNYYFTTNKKGNITASGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRTDEWKAIVKDFDTYETSEEPYISETAPHYHLENOKIGIRFRNDNDEIWPSLKTNGENNEKRKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDIYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMAEEAKRKQKEMVKDTKKLLATLEKQTQGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETXVQGWKNGFNLPRGIFTEPIREWFKRHQNDSEEYEKVETLDRVGLVTKVIPLFFKKEDSKDKEEYLKKDAQKEINNCVQPFYGFPYNVGNIHKPDEKDFLPSEERKKLWGDKKYKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSETELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKTLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETKE (SEQ ID  NO: 113) PIN17_0200AFJ07523 MKMEDDKKTKESTNMLDNKFLFWAAFLNLARHNVYITVNHINKVLEL [PrevotellaKNKKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLE intermediaTAIYTKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQEARNYYS 17]HYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEEFNYYFTTNKKGNITASGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRTDEWKAIVKDFDTYETSEEPYISETAPHYHLENQKIGIRFRNDNDEIWPSLKTNGENNEKRKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDIYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMAEEAKRKQKEMVKDTKKLLATLEKQTQGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSEEYEKVETLDRVGLVTKVIPLFFKKEDSKDKEEYLKKDAQKEINNCVQPFYGFPYNVGNIHKPDEKDFLPSEERKKLWGDKKYKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTYVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSETELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKTLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETKE (SEQ ID NO: 114)Prevotella BAU18623 MEDDKKTTDSISYELKDKFLFWAAFLNLARHNVYITVNHINKVLELKNintermedia KKDQDIIIDNDQDILAIKTHWEKVNGDLNKTERLRELMTKHFPFLETAIYSKNKEDKEEVKQEKQAKAQSFDSLKHCLFLFLEKLQETRNYYSHYKYSESTKEPMLEKELLKKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRTEEDFNYYFTRNKKGNITESGLLFFVSLFLEKKDAIWMQQKLRGFKDNRESKKKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEDREKFKVPFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTFHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKAIVKDLDTYETSNERYISETTPHYHLENQKIGIRFRNDNDEIWPSLKTNGENNEKSKYKLDKQYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDMYKLYDAFANGEINNIDDLEKYCEDKGIPKRHLPKQMVAILYDEHKDMVKEAKRKQRKMVKDTEKLLAALEKQTQEKTEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRSYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSKEYEKVEALDRVGLVTKVIPLFFKKEDSKDKEEDLKKDAQKEINNCVQPFYSFPYNVGNIHKPDEKDFLHREERIELWDKKKDKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDRIVTWLLCTELIDKLKVEGLNVEELKKLRLKDIDTDTAKQEKNNILNRVMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSEAELKSNPISKSLVEYELGEYQNARIETIKDMLLLEETLIEKYKNLPTDNFSDMLNGWLEGKDEADKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADTSEEKKLDIANQLKDKTHKIIKRIIEIEKPIETKE (SEQ ID NO: 115)HMPREF6485_0083 EFU31981MQKQDKLFVDRKKNAIFAFPKYITIMENKEKPEPIYYELTDKHFWAAF [PrevotellaLNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDK buccaeKVRLRDLIMKHFPFLEAAAYEMTNSKSPNNKEQREKEQSEALSLNNL ATCCKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANV 33574]RLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLFFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDTFSDDYNAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNOPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSAHNNLFPSLQTDKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTRRLQNTNILQGHLPKQMISILKGRQKDMGKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNRLKPKKRQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKALVKDRRLNGLFSFAETTDLNLEEHPISKLSVDLELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 116) HMPREF9144_1146EGQ18444 MKEEEKGKTPVVSTYNKDDKHFWAAFLNLARHNVYITVNHINKILGE [PrevotellaGEINRDGYENTLEKSWNEIKDINKKDRLSKLIIKHFPFLEVTTYQRNSA pallensDTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLER ATCCPKFEEDLQEKMYNIFDASIQLVKEDYKHNTDIKTEEDFKHLDRKGQFK 700821]YSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKIEGFKCSNESYQKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDRKKFRVPIEIADEDYDAEQEPFKNALVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIETKKKENKNDKQEKHKIEEIIENKITEIYALYDAFANGKINSIDKLEEYCKGKDIEIGHLPKQMIAILKSEHKDMATEAKRKQEEMLADVQKSLESLDNQINEEIENVERKNSSLKSGEIASWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIESSNPHPFLNNTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLMLKEPKTNCETLVQGWKNGFNLPRGIFTEPIRKWFMEHRKNITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYLFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFQSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKIIKEKNNILNRIMPMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTHSKAESKSNTISKSRVEYELGEYQKARIEIIKDMLALEETLIDKYNSLDTDNFHNMLTGWLKLKDEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKIIEIEKPIETKE (SEQ ID NO: 117) HMPREF9714_02132 EHO08761MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKR [MyroidesNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVD odoratimimusKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVI CCUGENKVLDFLNSSLVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAY 12901]KKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVVQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKANYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMTEEFKSKWKGYQHTELQKLFAYYDTSKSDLDLILSDMVMVKDYPIELIALVKKSRTLVDFLNKYLEARLGYMENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYQQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLOGLVPIGMDVREMLILSTDVKFIKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYTS (SEQ ID NO: 118) HMPREF9711_00870 EKB06014MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKR [MyroidesNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVD odoratimimusKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVI CCUGENKVLDFLNSSLVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAY 3837]KKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVVQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKANYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMTEEFKSKWKGYQHTELQKLFAYYDTSKSDLDLILSDMVMVKDYPIELIALVKKSRTLVDFLNKYLEARLGYMENVITRVKNSIGTPQFKTVRKECFTFLKKSNYTVVSLDKQIERILSMPLFIERGFMDDKPTMLEGKSYQQHKEDFADWFVHYKENSNYQNFYDTEVYEIITEDKREQAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSGYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECIVYNQVANKESLKQSGNENFKQYVLQGLLPRGTDVREMLILSTDVKFKKEEIMQLGQVREVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRPISDNEYYAEYYMEIFRSIKEKYAS (SEQ ID NO: 119) HMPREF9699_02005 EKB54193MENKTSLGNNIYYNPFKPQDKSYFAGYFNAAMENTDSVFRELGKRLK [BergeyellaGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEVPIKE zoohelcumRKENFKKNFKGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKST ATCCVLTVKKKKVKTDKTKEILKKSIEKQLDILCQKKLEYLRDTARKIEEKR 43767]RNQRERGEKELVAPFKYSDKRDDLIAAIYNDAFDVYIDKKKDSLKESSKAKYNTKSDPQQEEGIDLKIPISKNGVVFLLSLFLTKQETHAFKSKIAGFKATVIDEATVSEATVSHGKNSICFMATHEIFSHLAYKKLKRKVRTAEINYGEAENAEQLSVYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKENLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDREHYWEIFPNPNYDFPKENISVNDKDFPIAGSILDREKQPVAGKIGIKVKLLNQQYVSEVDKAVKAHQLKQRKASKPSIQNIIEEIVPINESNPKEAIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEKKEVGKIQAQIQQIIDKDTNAKILKPYQDGNSTAIDKEKLIKDLKQEQNILQKLKDEQTVREKEYNDFIAYQDKNREINKVRDRNHKQYLKDNLKRKYPEAPARKEVLYYREKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPEKVFQHLPFKLGGYFQQKYLYQFYTCYLDKRLEYISGINQQAENFKSENKVFKKVENECFKFLKKQNYTHKELDARVQSILGYPIFLERGFMDEKPTIIKGKTFKGNEALFADWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRKTKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDQFSLEDLYQSREERLGNQERARQTGERNTNYIWNKTVDLKLCDGKITVENVKLKNVGDFIKYEYDQRVQAFLKYEENIEWQAFLIKESKEEENYPYVVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTEPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKKEKEALIK (SEQ ID NO: 120) HMPREF9151_01387EKY00089 MMEKENVQGSHIYYEPTDKCFWAAFYNLARHNAYLTIAHINSFVNSK [PrevotellaKGINNDDKVLDIIDDWSKFDNDLLMGARLNKLILKHFPFLKAPLYQL saccharolyticaAKRKTRKQQGKEQQDYEKKGDEDPEVIQEAIANAFKMANVRKTLHA F0055]FLKQLEDLRNHFSHYNYNSPAKKMEVKFDDGFCNKLYYVFDAALQMVKDDNRMNPEINMQTDFEHLVRLGRNRKIPNTFKYNFTNSDGTINNNGLLFFVSLFLEKRDAIWMQKKIKGFKGGTENYMRMTNEVFCRNRMVIPKLRLETDYDNHQLMFDMLNELVRCPLSLYKRLKQEDQDKFRVPIEFLDEDNEADNPYQENANSDENPTEETDPLKNTLVRHQHRFPYFVLRYFDLNEVFKQLRFQINLGCYHFSIYDKTIGERTEKRHLTRTLFGFDRLQNFSVKLQPEHWKNMVKHLDTEESSDKPYLSDAMPHYQIENEKIGIHFLKTDTEKKETVWPSLEVEEVSSNRNKYKSEKNLTADAFLSTHELLPMMFYYQLLSSEEKTRAAAGDKVQGVLQSYRKKIFDIYDDFANGTINSMQKLDERLAKDNLLRGNMPQQMLAILEHQEPDMEQKAKEKLDRLITETKKRIGKLEDQFKQKVRIGKRRADLPKVGSIADWLVNDMMRFQPAKRNADNTGVPDSKANSTEYRLLQEALAFYSAYKDRLEPYFRQVNLIGGTNPHPFLHRVDWKKCNHLLSFYHDYLEAKEQYLSHLSPADWQKHQHFLLLKVRKDIQNEKKDWKKSLVAGWKNGFNLPRGLFTESIKTWTSTDADKVQITDTKLFENRVGLIAKLIPLYYDKVYNDKPQPFYQYPFNINDRYKPEDTRKRFTAASSKLWNEKKMLYKNAQPDSSDKIEYPQYLDFLSWKKLERELRMLRNQDMMVWLMCKDLFAQCTVEGVEFADLKLSQLEVDVNVQDNLNVLNNVSSMILPLSVYPSDAQGNVLRNSTSKPLHTVYVQENNTKLLKQGNFKSLLKDRRLNGLFSFIAAEGEDLQQHPLTKNRLEYELSIYQTMRISVFEQTLQLEKAILTRNKTLCGNNFNNLLNSWSEHRTDKKTLQPDIDFLIAVRNAFSHNQYPMSTNTVMQGIEKFNIQTPKLEEKDGLGIASQLAKKTKDAASRLQNIINGGTN (SEQ ID NO: 121) A343_1752 EOA10535MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLTHINDRQLAYS [PorphyromonasKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGK gingivalisKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLK JCVIDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHN SC001]DKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRTSLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAERVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFAGIENASRENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDETLFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMVERIIQA (SEQ ID  NO: 122)HMPREF1981_03090 ERI81700MESIKNSQKSTGKTLQKDPPYFGLYLNMALLNVRKVENFIIRKWLGD [BacteroidesVALLPEKSGFHSLLTTDNLSSAKWTRFYYKSRKFLPFLEMFDSDKKSY pyogenesENRRETTECLDTIDRQKISSLLKEVYGKLQDIRNAFSHYHIDDQSVKHT F0041]ALIISSEMHRFIENAYSFALQKTRARFTGVFVETDFLQAEEKGDNKKFFAIGGNEGIKLKDNALIFLICLFLDREEAFKFLSRATGFKSTKEKGFLAVRETFCALCCRQPHERLLSVNPREALLMDMLNELNRCPDILFEMLDEKDQKSFLPLLGEEEQAHILENSLNDELCEAIDDPFEMTASLSKRYRYKNRFPYLMLRYIEEKNLLPFIRFRIDLGCLELASYPKKMGEENNYERSVTDHAMAFGRLTDFHNEDAVLOQITKGITDEVRFSLYAPRYAIYNNKIGFVRTGGSDKISFPTLKKKGGEGHCVAYTLQNTKSFGFISIYDLRKILLLSFLDKDKAKNIVSGLLEQCEKHWKDLSENLFDAIRTELQKEFPVPLIRYTLPRSKGGKLVSSKLADKQEKYESEFERRKEKLTEILSEKDFDLSQIPRRMIDEWLNVLPTSREKKLKGYVETLKLDCRERLRVFEKREKGEHPVPPRIGEMATDLAKDIIRMVIDQGVKQRITSAYYSEIQRCLAQYAGDDNRRHLDSIIRELRLKDTKNGHPFLGKVLRPGLGHTEKLYQRYFEEKKEWLEATFYPAASPKILVPRFVNPPTGKQKELPLIIRNLMKERPEWRDWKQRKNSHPIDLPSQLFENEICRLLKDKIGKEPSGKLKWNEMFKLYWDKEFPNGMQRFYRCKRRVEVFDKVVEYEYSEEGGNYKKYYEALIDEVVRQKISSSKEKSKLQVEDLTLSVRRVFKRAINEKEYQLRLLCEDDRLLFMAVRDLYDWKEAQLDLDKIDNMLGEPVSVSQVIQLEGGQPDAVIKAECKLKDVSKLMRYCYDGRVKGLMPYFANHEATQEQVEMELRHYEDHRRRVFNWVFALEKSVLKNEKLRRFYEESQGGCEHRRCIDALRKASLVSEEEYEFLVFHIRNKSAHNQFPDLEIGKLPPNVTSGFCECIWSKYKAIICRIIPFIDPERRFFGKLLEQK (SEQ ID NO: 123) HMPREF1553_02065 ERJ65637MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESFIVRIKFGK [PorphyromonasKKLNEESLKQSLLCDHLLSVDRWTKWGHSRRYLPFLHYFDPDSQIE gingivalisKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVS F0568]PDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMSGEASYPVTLFSLFAPRYALYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRFTQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 124)HMPREF1988_01768 ERJ81987MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESFIVRIKFGK [PorphyromonasKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIE gingivalisKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVS F0185]PDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMSGEASYPVTLFSLFAPRYALYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRFTQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 125)HMPREF1990_01800 ERJ87335MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESFIVRIKFGK [PorphyromonasKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIE gingivalisKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVS W4087]PDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMSGEASYPVTLFSLFAPRYALYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRFTQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKIMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEIPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 126) M573_117042KJJ86756 MKMEDDKKTTESTNMLDNKHFWAAFLNLARHNVYITVNHINKVLEL [PrevotellaKNKKDQDIIIDNDQILAIKTHWEKVNGDLNKTERLRELMTKHFPFLE intermediaTAIYTKNKEDKEEVKQEKQAEAQSLESLKDCLFLFLEKLQEARNYYS ZT]HYKYSESTKEPMLEEGLLEKMYNIFDDNIQLVIKDYQHNKDINPDEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWMQQKLTGFKDNRESKKKMTHEVFCRRRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYERLQGEYRKKFNVPFDSADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKLIGGQKEDRHLTHKLYGFERIQEFAKQNRPDEWKALVKDLDTYETSNERYISETTPHYHLENQKIGIRFRNGNKEIWPSLKTNGENNEKSKYKLDKPYQAEAFLSVHELLPMMFYYLLLKKEEPNNDKKNASIVEGFIKREIRDMYKLYDAFANGEINNIGDLEKYCEDKGIPKRHLPKQMVAILYDEPKDMVKEAKRKQKEMVKDTKKLLATLEKQTQEEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLINSSNPHPFLKWTKWEECNNILSFYRNYLTKKIEFLNKLKPEDWEKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFKRHQNDSKEYEKVEALKRVGLVTKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPDEKDFLPSEERKKLWGDKKDKFKGYKAKVKSKKLTDKEKEEYRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKMKVEGLNVEELQKLRLKDIDTDTAKQEKNNILNRIMPMQLPVTVYEIDDSHNIVKDRPLHTVYIEETKTKLLKQGNFKALVKDRRLNGLFSFVDTSSKAELKDKPISKSVVEYELGEYQNARIETIKDMLLLEKTLIKKYEKLPTDNFSDMLNGWLEGKDESDKARFQNDVKLLVAVRNAFSHNQYPMRNRIAFANINPFSLSSADISEEKKLDIANQLKDKTHKIIKKIIEIEKPIETKE (SEQ ID NO: 127) A2033_10205OFX18020.1 MENQTQKGKGIYYYYTKNEDKHYFGSFLNLANNNIEQIIEEFRIRLSL[Bacteroidetes KDEKNIKEIINNYFTDKKSYTDWERGINILKEYLPVIDYLDLAITDKEFbacterium EKIDLKQKETAKRKYFRTNFSLLIDTIIDLRNFYTHYFHKPISINPDVAK GWA2_31_9FLDKNLLNVCLDIKKQKMKTDKTKQALKDGLDKELKKLIELKKAELKEKKIKTWNITENVEGAVYNDAFNHMVYKNNAGVTILKDYHKSILPDDKIDSELKLNFSISGLVFLLSMFLSKKEIEQFKSNLEGFKGKVIGENGEYEISKFNNSLKYMATHWIFSYLTFKGLKQRVKNTFDKETLLMQMIDELNKVPHEVYQTLSKEQQNEFLEDINEYVQDNEENKKSMENSIVVHPVIRKRYDDKFNYFAIRFLDEFANFPTLKFFVTAGNFVHDKREKQIQGSMLTSDRMIKEKINVFGKLTEIAKYKSDYFSNENTLETSEWELFPNPSYLLIQNNIPVHIDLIHNTEEAKQCQIAIDRIKCTTNPAKKRNTRKSKEEIIKIIYQKNKNIKYGDPTALLSSNELPALIYELLVNKKSGKELENIIVEKIVNQYKTIAGFEKGQNLSNSLITKKLKKSEPNEDKINAEKIILAINRELEITENKLNIIKNNRAEFRTGAKRKHIFYSKELGQEATWIAYDLKRFMPEASRKEWKGFHHSELQKFLAFYDRNKNDAKALLNMFWNFDNDQLIGNDLNSAFREFHFDKFYEKYLIKRDEILEGFKSFISNFKDEPKLLKKGIKDIYRVFDKRYYIIKSTNAQKEQLLSKPICLPRGIFDNKPTYIEGVKVESNSALFADWYQYTYSDKHEFQSFYDMTPRDYKEQFEKFELNNIKSIQNKKNLNKSDKFIYFRYKQDLKIKQIKSQDLFIKLMVDELFNVVFKNNIELNLKKLYQTSDERFKNQLIADVQKNREKGDTSDNKMNENFIWNMTIPLSLCNGQIEEPKVKLKDIGKFRKLETDDKVIQLLEYDKSKVWKKLEIEDELENMPNSYERIRREKLLKGIQEFEHFLLEKEKFDGINHPKHFEQDLNPNFKTYVINGVLRKNSKLNYTEIDKLLDLEHISIKDIETSAKEIHLAYFLIHVRNKFGHNQLPKLEAFELMKKYYKKNNEETYAEYFHKVSSQIVNEFKNSLEK HS (SEQ ID NO: 128)SAMN05421542_0666 SDI27289.1MEKTQTGLGIYYDHTKLQDKYFFGGFFNLAQNNIDNVIKAFIIKFFPER [ChryseobacteriumKDKDINIAQFLDICFKDNDADSDFQKKNKFLRIHFPVIGFLISDNDKAG jejuense]FKKKFALLLKTISELRNFYTHYYHKSIEFPSELFELLDDIFVKTTSEIKKLKKKDDKTQQLLNKNLSEEYDIRYQQQIERLKELKAQGKRVSLTDETAIRNGVFNAAFNHLIYRDGENVKPSRLYQSSYSEPDPAENGISLSQNSILFLLSMFLERKETEDLKSRVKGFKAKIIKQGEEQISGLKFMATHWVFSYLCFKGIKQKLSTEFHEETLLIQIIDELSKVPDEVYSAFDSKTKEKFLEDINEYMKEGNADLSLEDSKVIHPVIRKRYENKFNYFAIRFLDEYLSSTSLKFQVHVGNYVHDRRVKHINGTGFQTERIVKDRIKVFGRLSNISNLKADYIKEQLELPNDSNGWEIFPNPSYIFIDNNVPIHVLADEATKKGIELFKDKRRKEQPEELQKRKGKISKYNIVSMIYKEAKGKDKLRIDEPLALLSLNEIPALLYQILEKGATPKDIELIIKNKLTERFEKIKNYDPETPAPASQLSKRLRNNTTAKGQEALNAEKLSLLIEREIENTETKLSSIEEKRLKAKKEQRRNTPQRSIFSNSDLGRIAAWLADDIKRFMPAEQRKNWKGYQHSQLQQSLAYFEKRPQEAFLLLKEGWDTSDGSSYWNNWVMNSFLENNHFEKFYKNYLMKRVKYFSELAGNIKQHTHNTKFLRKFIKQQMPADLFPKRHYILKDLETEKNKVLSKPLVFSRGLFDNNPTFIKGVKVTENPELFAEWYSYGYKTEHVFQHFYGWERDYNELLDSELQKGNSFAKNSIYYNRESQLDLIKLKQDLKIKKIKIQDLFLKRIAEKLFENVFNYPTTLSLDEFYLTQEERAEKERIALAQSLREEGDNSPNIIKDDFIWSKTIAFRSKQIYEPAIKLKDIGKFNRFVLDDEESKASKLLSYDKNKIWNKEQLERELSIGENSYEVIRREKLFKEIQNLELQILSNWSWDGINHPREFEMEDQKNTRHPNFKMYLVNGILRKNINLYKEDEDFWLESLKENDFKTLPSEVLETKSEMVQLLFIVILIRNQFAHNQLPEIQFYNFIRKNYPEIQNNTVAELYLNLIKLAVQKLKDNS (SEQ ID NO: 129)SAMN05444360_11366 SHM52812.1MNTRVTGMGVSYDHTKKEDKHFFGGFLNLAQDNITAVIKAFCIKFDK [ChryseobacteriumNPMSSVQFAESCFTDKDSDTDFQNKVRYVRTHLPVIGYLNYGGDRNT carnipullorum]FRQKLSTLLKAVDSLRNFYTHYYHSPLALSTELFELLDTVFASVAVEVKQHKMKDDKTRQLLSKSLAEELDIRYKQQLERLKELKEQGKNIDLRDEAGIRNGVLNAAFNHLIYKEGEIAKPTLSYSSFYYGADSAENGITISQSGLLFLLSMFLGKKEIEDLKSIRIRGFKAKIVRDGEENISGLKFMATHWIFSYLSFKGMKQRLSTDFHEETLLIQIIDELSKVPDEVYHDFDTATREKFVEDINEYIREGNEDFSLGDSTIIHPVIRKRYENKFNYFAVRFLDEHKFPSLRFQVHLGNFVHDRRIKDIHGTGFQTERVVKDRIKVFGKLSEISSIKTEYIEKELDLDSDTGWEIFPNPSYVFIDNNIPIYISTNKTFKNGSSEFIKLRRKEKPEEMKMRGEDKKEKRDIASMIGNAGSLNSKTPLAMLSLNEMPALLYEILVKKTTPEEIELIIKEKLDSHFENIKNYDPEKPLPASQISKRLRNNTTDKGKKVINPEKLIHLINKEIDATEAKFALLAKNRKELKEKFRGKPLRQTIFSNMELGREATWLADDIKRFMPDILRKNWKGYQHNQLQQSLAFFNSRPKEAFTILQDGWDFADGSSFWNGWIINSFVKNRSFEYFYEAYFEGRKEYFSSLAENIKQHTSNHRNLRRFIDQQMPKGLFENRHYLLENLETEKNKILSKPLVFPRGLFDTKPTFIKGIKVDEQPELFAEWYQYGYSTEHVFQNFYGWERDYNDLLESELEKDNDFSKNSIHYSRTSQLELIKLKQDLKIKKIKIQDLFLKLIAGHIFENIFKYPASFSLDELYLTQEERLNKEQEALIQSQRKEGDHSDNIIKDNFIGSKTVTYESKQISEPNVKLKDIGKFNRLLDDKVKTLLSYNEDKVWNKNDLDLELSIGENSYEVIRREKLFKKIQNFELQTLTDWPWNGTDHPEEFGTTDNKGVNHPNFKMYVVNGILRKEITDWFKEGEDNWLENLNETHFKNLSFQELETKSKSIQTAFLIIMIRNQFAHNQLPAVQFFEFIQKKYPEIQGSTTSELYLNFINLAVVELLELLEK (SEQ ID  NO: 130)SAMN05421786_1011119 S1S70481.1METQILGNGISYDHTKTEDKHFFGGFLNTAQNNIDLLIKAYISKFESSP [ChryseobacteriumRKLNSVQFPDVCFKKNDSDADFQHKLQFIRKHLPVIQYLKYGGNREV ureilyticum]LKEKFRLLLQAVDSLRNFYTHFYHKPIQLPNELLTLLDTIFGEIGNEVRQNKMKDDKTRHLLKKNLSEELDFRYQEQLERLRKLKSEGKKVDLRDTEAIRNGVLNAAFNHLIFKDAEDFKPTVSYSSYYYDSDTAENGISISQSGLLFLLSMFLGRREMEDLKSRVRGFKARIIKHEEQHVSGLKFMATHWVFSEFCFKGIKTRLNADYHEETLLIQLIDELSKVPDELYRSFDVATRERFIEDINEYIRDGKEDKSLIESKIVHPVIRKRYESKFNYFAIRFLDEFVNFPTLRFQVHAGNYVHDRRIKSIEGTGFKTERLVKDRIKVFGKLSTISSLKAEYLAKAVNITDDTGWELLPHPSYVFIDNNIPIHLTVDPSFKNGVKEYQEKRKLQKPEEMKNRQGGDKMHKPAISSKIGKSKDINPESPVALLSMNEIPALLYEILVKKASPEEVEAKIRQKLTAVFERIRDYDPKVPLPASQVSKRLRNNTDTLSYNKEKLVELANKEVEQTERKLALITKNRRECREKVKGKFKRQKVFKNAELGTEATWLANDIKRFMPEEQKKNWKGYQHSQLQQSLAFFESRPGEARSLLQAGWDFSDGSSFWNGWVMNSFARDNTFDGFYESYLNGRMKYFLRLADNIAQQSSTNKLISNFIKQQMPKGLFDRRLYMLEDLATEKNKILSKPLIFPRGIFDDKPTFKKGVQVSEEPEAFADWYSYGYDVKHKFQEFYAWDRDYEELLREELEKDTAFTKNSIHYSRESQIELLAKKQDLKVKKVRIQDLYLKLMAEFLFENVFGHELALPLDQFYLTQEERLKQEQEAIVQSQRPKGDDSPNIVKENFIWSKTIPFKSGRVFEPNVKLKDIGKFRNLLTDEKVDILLSYNNTEIGKQVIENELIIGAGSYEFIRREQLFKEIQQMKRLSLRSVRGMGVPIRLNLK (SEQ ID NO: 131) Prevotella WP_004343581MQKQDKLFVDRKKNAIFAFPKYITIMENQEKPEPIYYELTDKHFWAAF buccaeLNLARHNVYTTINHINRRLEIAELKDDGYMMDIKGSWNEQAKKLDKKVRLRDLEMKHFPFLEAAAYEITNSKSPNNKEQREKEQSEALSLNNLKNVLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFHYHFADKEGNMTIAGLLEFVSLFLDKKDAIWMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWMQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYDAEEEPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAQQNQPEVWRKLVKDLDYFEASQEPYIPKTAPHYHLENEKIGIKFCSTHNNLFPSLKTEKTCNGRSKENLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANGEINSIADLTCRLQKTNILQGHLPKQMISILEGRQKDMEKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKIADWLVNDMMRFQPVQKDQNNIPINNSKANSTEYRMLQRALALFGSENFRLKAYFNQMNLVGNDNPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNKLKPQKGQFLDKKERVELWQKNKELFKNYPSEKKKTDLAYLDFLSWKKFERELRLIKNQDIVTWLMFKELFNMATVEGLKIGEIHLRDIDTNTANEESNNILNRIMPNIKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKVLAKDRRLNGLLSFAETTDIDLEKNPITKLSVDHELIKYQTTRISIFEMTLGLEKKLINKYPTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSFINQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 132) PorphyromonasWP_005873511 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHNLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKILTSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLNKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 133) PorphyromonasWP_005874195 MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSIUSLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDIZ.LREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKLGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFVGIEYASWENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDESFREMLESWSDPLLDKWPDLQREVRLLIAVRNAFSHNQYPMYDETIFSSIRKYDPSSLDAIEERMGLNIAHRLSEEVKLAKEMVERIIQA (SEQ ID  NO: 134) PrevotellaWP_006044833 MKEEEKGKTPVVSTYNKDDKHFWAAFLNLARHNVYITVNHINKILGE pallensGEINIRDGYENTLEKSWNEIKDINKKDRLSKLIIKHFPFLEVTTYQRNSADTTKQKEEKQAEAQSLESLKKSFFVFIYKLRDLRNHYSHYKHSKSLERPKFEEDLQEKMYNIFDASIQLVKEDYKHNTDIKTEEDFKHLDRKGQFKYSFADNEGNITESGLLFFVSLFLEKKDAIWVQKKLEGFKCSNESYQKMTNEVFCRSRMLLPKLRLQSTQTQDWILLDMLNELIRCPKSLYERLREEDRKKFRVPIEIADEDYDAEQEPFKNALVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDYKESHHLTHKLYGFERIQEFTKQNRPDEWRKFVKTFNSFETSKEPYIPETTPHYHLENQKIGIRFRNDNDKIWPSLKTNSEKNEKSKYKLDKSFQAEAFLSVHELLPMMFYYLLLKTENTDNDNEIEFKKKENKNDKQEKHKIEEIIENKITEIYALYDAFANGKINSIDKLEEYCKGKDIEIGHLPKQMIAILKSEHKDMATEAKRKQEEMLADVQKSLESLDNQINEEIENVERKNSSLKSGEIASWLVNDMMRFQPVQKDNEGNPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIESSNPHPFLNNTEWEKCNNILSFYRSYLEAKKNFLESLKPEDWEKNQYFLMLKEPKTNCETLVQGWKNGFNLPRGIFTEPIRKWFMEHRKNITVAELKRVGLVAKVIPLFFSEEYKDSVQPFYNYLFNVGNINKPDEKNFLNCEERRELLRKKKDEFKKMTDKEKEENPSYLEFQSWNKFERELRLVRNQDIVTWLLCMELFNKKKIKELNVEKIYLKNINTNTTKKEKNTEEKNGEEKIIKEKNNILNIRIMPIMRLPIKVYGRENFSKNKKKKIRRNTFFTVYIEEKGTKLLKQGNFKALERDRRLGGLFSFVKTHSKAESKSNTISKSRVEYELGEYQKARIEIIKDMLALEETLIDKYNSLDTDNFHNMLMTGWLKLKDEPDKASFQNDVDLLIAVRNAFSHNQYPMRNRIAFANINPFSLSSANTSEEKGLGIANQLKDKTHKTIEKHEIEKPIETKE (SEQ ID NO: 135) Myroides odoratimimusWP_006261414 MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKRNTFGKLAKRDNGNLKNYIIHVEKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVIENKVLDFLNSSEVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFELTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRENYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKANYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLNFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMTEEFKSKWKGYQHTELQKLFAYYDTSKSDLDLILSDMVIMVKDYPIELIALVKKSRTLVDFLNKYLEARLGYMENVITRVKNSIGTPQEKTVRKECFTELKKSNYTVVSLDKQVERILSMPLFIERGFMDDKPTMLEGKSYQQHKEKFADWFVHYKENSNYQNFYDTEVYEITTEDKREKAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLSSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSAYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECSVYNQVANKESLKQSGNENFKQYVLQGLVPIGMDVREMLILSTDVKFIKEEIIQLGQAGEVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRSISDNEYYAEYYMEIFRSIKEKYTS (SEQ ID NO: 136) Myroides odoratimimus WP_006265509MKDILTTDTTEKQNRFYSHKIADKYFFGGYFNLASNNIYEVFEEVNKRNTFGKLAKRDNGNLKNYIIHVFKDELSISDFEKRVAIFASYFPILETVDKKSIKERNRTIDLTLSQRIRQFREMLISLVTAVDQLRNFYTHYHHSEIVIENKVLDFLNSSLVSTALHVKDKYLKTDKTKEFLKETIAAELDILIEAYKKKQIEKKNTRFKANKREDILNAIYNEAFWSFINDKDKDKETVVAKGADAYFEKNHHKSNDPDFALNISEKGIVYLLSFFLTNKEMDSLKANLTGFKGKVDRESGNSIKYMATQRIYSFHTYRGLKQKIRTSEEGVKETLLMQMIDELSKVPNVVYQHLSTTQQNSFIEDWNEYYKDYEDDVETDDLSRVIHPVIRKRYEDRFNYFAIRFLDEFFDFPTLRFQVHLGDYVHDRRTKQLGKVESDRIIKEKVTVFARLKDINSAKASYFHSLEEQDKEELDNKWTLFPNPSYDFPKEHTLQHQGEQKNAGKIGIYVKLRDTQYKEKAALEEARKSLNPKERSATKASKYDIITQIIEANDNVKSEKPLVFTGQPIAYLSMNDIHSMLFSLLTDNAELKKTPEEVEAKLIDQIGKQINEILSKDTDTKILKKYKDNDLKETDTDKITRDLARDKEEIEKLILEQKQRADDYNYTSSTKFNIDKSRKRKHLLFNAEKGKIGVWLANDIKRFMFKESKSKWKGYQHTELQKLFAYFDTSKSDLELILSDMVMVKDYPIELIDLVRKSRTLVDFLNKYLEARLGYIENVRTRVKNSIGTPQFKTVRKECFAFLKESNYWASLDKQIERILSMPLFIERGFMDSKPTMLEGKSYQQFIKEDFADWFVWKENSNYQNFYDTEVYEIITEDKREQAKVTKKIKQQQKNDVFTLMMVNYMLEEVLKLPSNDRLSLNELYQTKEERIVNKQVAKDTQERNKNYIWNKVVDLQLCEGLVRIDKVKLKDIGNFRKYENDSRVKEFLTYQSDIVWSGYLSNEVDSNKLYVIERQLDNYESIRSKELLKEVQEIECIVYNQVANKESLKQSGNENFKQYVLQGLLPRGTDVREMLILSTDVKFKKEEIMQLGQVREVEQDLYSLIYIRNKFAHNQLPIKEFFDFCENNYRPISDNEYYAEYYMEIFRSIKEKYAS (SEQ ID NO: 137) Prevotella WP_007412163MQKQDKLFVDRKKNAIFAFPKYITIMENQEKPEPRYYELTDKHFWAAF sp. MSX73LNLARHNVYTTINHINRRLEIAELKDDGYMMGIKGSWNEQAKKLDKKVRLRDLMKHFPFLEAAAYERRNSKSPNNKEQREKEQSEALSLNNLKNYLFIFLEKLQVLRNYYSHYKYSEESPKPIFETSLLKNMYKVFDANVRLVKRDYMHHENIDMQRDFTHLNRKKQVGRTKNIIDSPNFFSYHFADKEGNMTIAGLLFFVSLFLDKKDARVMQKKLKGFKDGRNLREQMTNEVFCRSRISLPKLKLENVQTKDWTVIQLDMLNELVRCPKSLYERLREKDRESFKVPFDIFSDDYDAFFFPFKNTLVRHQDRFPYFVLRYFDLNEIFEQLRFQIDLGTYHFSIYNKRIGDEDEVRHLTHHLYGFARIQDFAPQNQPEEWRKLVKDLDHFETSQEPYISKTAPHYHLENEKIGIKFCSTHNNLFPSLKREKTCNGRSKFNLGTQFTAEAFLSVHELLPMMFYYLLLTKDYSRKESADKVEGIIRKEISNIYAIYDAFANNEINSIADLTCRLQKTNILQGHLPKQMISILEGRQKDMEKEAERKIGEMIDDTQRRLDLLCKQTNQKIRIGKRNAGLLKSGKQIADWLVSDMMRFQPVQKDTNNAPINNSKANSTEYRMLQHALALFGSESSRLKAYFRQMNLVGNANPHPFLAETQWEHQTNILSFYRNYLEARKKYLKGLKPQNWKQYQHFLILKVQKTNRNTLVTGWKNSFNLPRGIFTQPIREWFEKHNNSKRIYDQILSFDRVGFVAKAIPLYFAEEYKDNVQPFYDYPFNIGNKLKPQKGQFLDKKERVELWQKNKELFKNYPSEKNKTDLAYLDFLSWKKFERELRLIKNQDRVLWLMFKELFKLTRVEGLKIGEIHLRDIDTNTANEESNNILNRIMPMKLPVKTYETDNKGNILKERPLATFYIEETETKVLKQGNFKVLAKDRRLNGLLSFAETTDIDLEKNPITKLSVDYELIKYQTTRISIFEMTLGLEKKLIDKYSTLPTDSFRNMLERWLQCKANRPELKNYVNSLIAVRNAFSHNQYPMYDATLFAEVKKFTLFPSVDTKKIELNIAPQLLEIVGKAIKEIEKSENKN (SEQ ID NO: 138) PorphyromonasWP_012458414 MTEQNERPYNGTYYTLEDKHFWAAFFNLARFINAYITLAHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKFIFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKTHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGIYHFARYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYRRQTTPFFYHIEKGKLGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSDEASAERVQGRIKRVIEDVYAVYDAFARGEINTRDELDACLADKGIRRGHLPRQMIGILSQEHKDMEEKVRKKLQEMPVDTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWRQKFERELRLVKNQDIITWMICRDLMEENKVEGLDTGTLYLKDIRTDVQEQGNLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYLEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHGNVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 139) PaludibacterWP_013446107 MKTSANNIYFNGINSFKKIFDSKGAIAPIAEKSCRNFDIKAQNDVNKEQpropionicigenes RIHYFAVGHTFKQLDTENLFEYVLDENLRAKRPTRFISLQQFDKEFIENIKRLISDIRNINSHYIHRFDPLKIDAVPTNIIDFLKESFELAVIQIYLKEKGINYLQFSENPHADQKLVAFLHDKFLPLDEKKTSMLQNETPQLKEYKEYRKYFKTLSKQAAIDQLLFAEKETDYIWNLFDSHPVLTISAGKYLSFYSCLFLLSMFLYKSEANQLISKIKGFKKNTTEEEKSKREIFTFFSKRFNSMDIDSEENQLVKFRDLILYLNHYPVAWNKDLELDSSNPAMTDKLKSKIIELEINRSFPLYEGNERFATFAKYQIWGKKHLGKSIEKEYINASFTDEEITAYTYETDTCPELKDAFIKKLADLKAAKGLFGKRKEKNESDIKKTETSIRELQHEPNPIKDKLIQRIEKNLLTVSYGRNQDRFMDFSARFLAEINYFGQDASFKMYHFYATDEQNSELEKYELPKDKKKYDSLKFHQGKLVHFISYKEHLKRYESWDDAFVIENNAIQLKLSFDGVENTVTIQRALLIYLLEDALRNIQNNTAENAGKQLLQEYYSHNKADLSAFKQILTQQDSIEPQQKTEFKKLLPRRLLNNYSPAINHLQTPHSSLPLILEKALLAEKRYCSLVVKAKAEGNYDDFTKRNKGKQFKLQFIRKAWNLMYFRNSYLQNVQAAGHHKSFHIERDEFNDFSRYMFAFEELSQYKYYLNEMFEKKGFFENNEFKILFQSGTSLENLYEKTKQKFEIWLASNTAKTNKPDNYHLNNYEQQFSNQLFFINLSHFINYLKSTGKLQTDANGQIIYEALNNVQYLIPEYYYTDKPERSESKSGNKLYNKLKATKLEDALLYEMAMCYLKADKQIADKAKHPITKLLTSDVEFNITNKEGIQLYHLLVPFKKIDAFIGLKMFIKEQQDKKHPTSFLANIVNYLELVKNDKDIRKTYEAFSTNPVKRTLTYDDLAKIDGHLISKSIKFTNVTLELERYFIFKESLIVKKGNNIDFKYIKGLRNYYNNEKKKNEGIRNKAFHFGIPDSKSYDQLIRDAEVMFIANEVKPTHATKYTDLNKQLHTVCDKLMETVHNDYFSKEGDGKKKREAAGQKYFENIISAK (SEQ ID NO: 140)Porphyromonas WP_013816155MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEEDTDGAEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFQIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRFTAEAFLSAHELMPMMFYYFLLREKYSEEASAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIGILSQEHKDMEEKIRKKLQEMMADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDWVQPFYNYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMICGDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRCPHLPDKNFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSFPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 141) FlavobacteriumWP_014165541 MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKEFKTR columnareINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLMSFFLHRKEFQVFTSGLEGFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLSLVTEYKKNVYLKETSNIDLSRFPLFPNPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNKKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLIKTINTDSSVTFENQPIDIPRLKNAIQKELTLTQEKLLNVKEHEIEVDNYNRNKNTYKFKNQPKNKVDDKKLQRKYVFYRNEIRQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKGLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGLIGLPPKILKKELSKRFKYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDIVERDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKAEREKIKADAKAYQKRNDSSLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKELLKQVQELEKQILEEYTDFLSTQIHPADFEREGNPNFKKYLAHSILENEDDLDKLPEKVEAMRELDETITNPIIKKAIVLIIIRNKMAFINQYPPKFIYDLANRFVPKKEEEYFATYFNRVFETITKELWENKEKKDKTQV (SEQ ID NO: 142) Psychroflexus WP_015024765MESIIGLGLSFNPYKTADKHYFGSFLNLVENNLNAVFAEFKERISYKA torquisKDENISSLIEKHFIDNMSIVDYEKKISILNGYLPIIDFLDDELENNLNTRVKNFKKNFIILAEAIEKLRDYYTHFYHDPITFEDNKEPLLELLDEVLLKTILDVKKKYLKTDKTKEILKDSLREEMDLLVIRKTDELREKKKTNPKIQHTDSSQIKNSIFNDAFQGLLYEDKGNNKKTQVSHRAKTRLNPKDIHKQEERDFEIPLSTSGLVFLMSLFLSKKEIEDFKSNIKGFKGKVVKDENHNSLKYMATHRVYSILAFKGLKYRIKTDTFSKETLMMQMIDELSKVPDCVYQNLSETKQKDFIEDWNEYFKDNEENTENLENSRVVHPVIRKRYEDKFNYFAIRFLDEFANFKTLKFQVFMGYYIHDQRTKTIGTTNITTERTVKEKINVFGKLSKMDNLKKHFFSQLSDDENTDWEFFPNPSYNFLTQADNSPANNIPIYLELKNQQIIKEKDAIKAEVNQTQNRNPNKPSKRDLLNKILKTYEDFHQGDPTAILSLNEIPALLHLFLVKPNNKTGQQIENIIRIKIEKQFKAINHPSKNNKGIPKSLFADTNVRVNAIKLKKDLEAELDMLNKKHIAFKENQKASSNYDKLLKEHQFTPKNKRPELRKYVFYKSEKGEEATWLANDIKRFMPKDFKTKWKGCQHSELQRKLAFYDRHTKQDIKELLSGCEFDHSLLDINAYFQKDNFEDFFSKYLENRIETLEGVLKKLHDFKNEPTPLKGVFKNCFKFLKRQNYVTESPEIIKKRTLAKPTFLPRGVFDERPTMKKGKNPLKDKNEFAEWFVEYLENKDYQKFYNAEEYRMRDADFKKNAVIKKQKLKDFYTLQMVNYLLKEVFGKDEMNLQLSELFQTRQERLKLQGIAKKQMNKETGDSSENTRNQTYIWRNKDVPVSFFNGKVTIDKVKLKNIGKYKRYEIIDERVKTFIGYEVDEKWMMYLPHNVVKDRYSVKPINVIDLQIQEYEEIRSHELLKEIQNLEQYIYDHTTDKNILLQDGNPNFKMYVLNGLLIGIKQVNIPDFIVLKQNTNFDKIDFTGIASCSELEKKTIILIAIRNKFAHNQLPNKMIYDLANEFLKIEKNETYANYYLKVLKKMISDLA (SEQ ID NO: 143) RiemerellaWP_015345620 MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHLanatipestifer NRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKHQPWEPYQYCLLLKVPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKFSGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKKEEHYEYWQQNKPQSPTESQRLEMTSDRWKDYLLYKRWQHLEKKLRLYRNQDIMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPTTAFGEVQYHETPIRTVYIREEQTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHASLSSLENEFRTLLEEWKKKYAASSMVTDKHIAFIASVRNAFCHNQYPFYKETLHAPILLFTVAQPTTEEKDGLGIAEALLKVLREYCEIVKSQI (SEQ ID NO: 144)Prevotella WP_021584635 MENDKRLEESACYTLNDKHFWAAFLNLARHNVYITVNHINKTLELKpleuritidis NKKNQEIIIDNDQDILAIKTHWAKVNGDLNKTDRLRELMIKFSFPFLEAAIYSNNKEDKEEVKEEKQAKAQSFKSLKDCLFLFLEKLQEARNYYSHYKYSESSKEPEFEEGLLEKMYNTFDASIRLVKEDYQYNKDIDPEKDFKHLERKEDFNYLFTDKDNKGKITKNGLLFFVSLFLEKKDAIWMQQKFRGFKDNRGNKEKMTHEVFCRSRMLLPKIRLESTQTQDWILLDMLNELIRCPKSLYERLQGAYREKFKVPFDSIDEDYDAEQEPFRNTLVRHQDRFPYFALRYFDYNEIFKNLRFQIDLGTYHFSIYKKLTGGKKEDRHLTHKLYGFERIQEFTKQNRPDKWQAIIKDLDTYETSNERYISETTPHYHLENQKIGIRFRNDNNDIWPSLKTNGEKNEKSKYNLDKPYQAEAFLSVHELLPMMFYYLLLKMENTDNDKEDNEVGTKKKGNKNNKQEKHKIEEIIENKIKDIYALYDAFTNGEINSDELAEQREGKDIEIGHLPKQLIVILKNKSKDMAEKANRKQKEMIKDTKKRLATLDKQVKGEIEDGGRNIRLLKSGEIARWLVNDMMRFQPVQKDNEGKPLNNSKANSTEYQMLQRSLALYNKEEKPTRYFRQVNLIKSSNPHPFLEDTKWEECYNILSFYRNYLKAKIKFLNKLKPEDWKKNQYFLMLKEPKTNRKTLVQGWKNGFNLPRGIFTEPIKEWFKRHQNDSEEYKKVEALDRVGLVAKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPEEKNFLHCEERRKLWDKKKDKFKGYKAKEKSKKMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDILTWLLCTKLIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRVMPMRLPVTVYEIDKSFNIVKDKPLHTVYIEETGTKLLKQGNFKALVKDRRLNGLFSFVKTSSEAESKSKPISKLRVEYELGAYQKARIDIIKDMLALEKTLIDNDENLPTNKFSDMLKSWLKGKGEANKARLQNDVGLLVAVRNAFSHNQYPMYNSEVFKGMKLLSLSSDIPEKEGLGIAKQLKDKIKETIERIIEIEKEIRN (SEQ ID NO: 145)Porphyromonas WP_021663197MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQPvLIRELYSLLDFLRNDFSFINRLDGTTFEHLEVSPDISSFITGTYSLACGELAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPFIDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASFISVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFFTRAVNEREFMLRLVQEDDRLMLMATNKMMTDREEDTLPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 146) PorphyromonasWP_021665475 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGFISRRYLPFLHYFDPDSQIEKDFTDSKTGVDPDSAQRLIRELYSLLDFLRNDFSFFNRLDGTTFEHLEVSPDISSFITG1YSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTYSGLAFFICLFLDREQASGMLSRIRGFKRTNENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDFSALAFGKLSDFQNEEEVSRMTSGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAFGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLTTSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFYPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYTRYRYDRRVPGLMSFTFPEFTKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 147) PorphyromonasWP_021677657 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKWGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEFSLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRTLDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 148) PorphyromonasWP_021680012 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMDQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLQKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRHQFRAIVAELRLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKVMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKIMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEIPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 149) PorphyromonasWP_023846767 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPRSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEFHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 150) PrevotellaWP_036884929 MKNDNNSTKSTDYTLGDKHFWAAFLNLARHNVYITVNHINKVLELK falseniiNKKDQEIIIDNDQDILAIKTLWGKVDTDINKKDRLRELIMKHFPFLEAATYQQSSTNNTKQKEEEQAKAQSFESLKDCLFLFLEKLREARNYYSHYKHSKSLEEPKLEEKLLENMYNIFDTNVQLVIKDYEHNKDINPEEDFKHLGRAEGEFNYYFTRNKKGNITESGLLFFVSLFLEKKDAIWAQTKIKGFKDNRENKQKMTHEVFCRSRMLLPKLRLESTQTQDWILLDMLNELIRCPKSLYKRLQGEKREKFRVPFDPADEDYDAEQEPFKNTLVRHQDRFPYFALRYFDYNEIFTNLRFQIDLGTYHFSIYKKQIGDKKEDRHLTHKLYGFERIQEFAKENRPDEWKALVKDLDTFEESNEPYISETTPHYHLENQKIGIRNKNKKKKKTIWPSLETKTTVNERSKYNLGKSFKAEAFLSVHELLPMMFYYLLLNKEEPNNGKINASKVEGIIEKKIRDIYKLYGAFANEEINNEEELKEYCEGKDIAIRHLPKQMIAILKNEYKDMAKKAEDKQKKMIKDTKKRLAALDKQVKGEVEDGGRNIKPLKSGRIASWLVNDMMRFQPVQRDRDGYPLNNSKANSTEYQLLQRTLALFGSERERLAPYFRQMNLIGKDNPHPFLKDTKWKEHNNILSFYRSYLEAKKNFLGSLKPEDWKKNQYFLKLKEPKTNRETLVQGWKNGFNLPRGIFTEPIREWFIRHQNESEEYKKVKDFDRIGLVAKVIPLFFKEDYQKEIEDYVQPFYGYPFNVGNIHNSQEGTFLNKKEREELWKGNKTKFKDYKTKEKNKEKTNKDKFKKKTDEEKEEFRSYLDFQSWKKFERELRLVRNQDIVTWLLCMELIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRIMPMELPVTVYETDDSNNIIKDKPLHTIYIKEAETKLLKQGNFKALVKDRRLNGLFSFVETSSEAELKSKPISKSLVEYELGEYQRARVEIIKDMLRLEETLIGNDEKLPTNKFRQMLDKWLEHKKETDDTDLKNDVKLLTEVRNAFSHNQYPMRDRIAFANIKPFSLSSANTSNEEGLGIAKKLKDKTKETIDRIIEIEEQTATKR (SEQ ID NO: 151) PrevotellaWP_036931485 MENDKRLEESTCYTLNDKHFWAAFLNLARHNVYITINHINKLLEIRQIpleuritidis DNDEKVLDIKALWQKVDKDINQKARLRELMIKHFPFLEAAIYSNNKEDKEEVKEEKQAKAQSFKSLKQCLFLFLEKLQEARNYYSHYKSSESSKEPEFEEGLLEKMYNTFGVSIRLVKEDYQYNKDIDPEKDFKHLERKEDFNYLFTDKDNKGKITKNGLLFFVSLFLEKKDAIWMQQKLRGFKDNRGNKEKMTHEVFCRSRMLLPKIRLESTQTQDWILLDMLNELIRCPKSLYERLQGAYREKFKVPFDSIDEDYDAEQEPFRNTLVRHQDRFPYFALRYFDYNEIFKNLRFQIDLGTYHFSIYKKLIGDNKEDRHLTHKLYGFERIQEFAKQKRPNEWQALVKDLDIYETSNEQYISETTPHYHLENQKIGIRFKNKKDKIWPSLETNGKENEKSKYNLDKSFQAEAFLSIHELLPMMFYDLLLKKEEPNNDEKNASIVEGFIKKEIKRMYAIYDAFANEEINSKEGLEEYCKNKGFQERHLPKQMIAILTNKSKNMAEKAKRKQKEMIKDTKKRLATLDKQVKGEIEDGGRNIRLLKSGEIARWLVNDMMRFQSVQKDKEGKPLNNSKANSTEYQMLQRSLALYNKEQKPTPYFIQVNLIKSSNPHPFLEETKWEECNNILSFYRSYLEAKKNFLESLKPEDWKKNQYFLMLKEPKTNRKTLVQGWKNGFNLPRGIFTEPIKEWFKRHQNDSEEYKKVEALDRVGLVAKVIPLFFKEEYFKEDAQKEINNCVQPFYSFPYNVGNIHKPEEKNFLHCEERRKLWDKKKDKFKGYKAKEKSKKMTDKEKEEHRSYLEFQSWNKFERELRLVRNQDIVTWLLCTELIDKLKIDELNIEELQKLRLKDIDTDTAKKEKNNILNRIMPMQLPVTVYEIDKSFNIVKDKPLHTIYIEETGTKLLKQGNFKALVKDRRLNGLFSFVKTSSEAESKSKPISKLRVEYELGAYQKARIDIIKDMLALEKTLIDNDENLPTNKFSDMLKSWLKGKGEANKARLQNDVDLLVAIRNAFSHNQYPMYNSEVFKGMKLLSLSSDIPEKEGLGIAKQLKDKIKETIERIIEIEKEIRN (SEQ ID NO: 152) [Porphyromonas WP_039417390MTEQNERPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGNNDNPFFKHHFVDREGTVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPIDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQV (SEQ ID NO: 153) PorphyromonasWP_039418912 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRMDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHDTRWESHTTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTNVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLATYYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDKNFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 154)Porphyromonas WP_039419792MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRARFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKVIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTRDELDACLADKGIRRGHLPKQMIGILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLDETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATYYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 155) PorphyromonasWP_039426176 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTGPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIKDVYAIYDAFARDEINTLKELDACSADKGIRRGHLPKQMIGILSQEHKNMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLDETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENRPFLLLKEPKNDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLNIEENKVEGLDTGTLYLKDIRTDVHEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDENFREMLESWSDPLLGKWPDLHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 156) PorphyromonasWP_039431778 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREDDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTSPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKDMEEKIRKKLOEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKKRLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQV (SEQ ID NO: 157)Porphyromonas WP_039437199MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDEDILFFKGQWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKFFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDEVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDGEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIGILSQERKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDESFREMLESWSDPLLTKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIWKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETIERIIQA (SEQ ID NO: 158) PorphyromonasWP_039442171 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNLDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSGSSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHYHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTGPYEQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRACFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYLETGDKPYISQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGTTRTGRSKCAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDTCLADKGIRRGHLPKQMITILSQERKDMKEKIRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDASGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRVENCPFLLLKEPKTDRQTLVAGWKDEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEDRAEEWERGMERFRDLEAWSHSAARRIKDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEAPLATVYIEERNTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLSRYPHLPDESFREMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQA (SEQ ID NO: 159)Porphyromonas WP_039445055MNTVPATENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gulaeKKLNEESLKQSLLCDHLLSIDRWTKVYGHSRRYLPFLHCFDPDSGIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLKVSPDISSFITGAYTFACERAQSRFADFFKPDDFLLAKNRKEQLISVADGKECLTVSGFAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMISGEASYPVRFSLFAPRYAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSDFLRKANRILDETAEGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVRDKKRELRTAGKPVPPDLAAYIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDEENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDHENRFFGKLLNNMSQPINDL (SEQ ID NO: 160) CapnocytophagaWP_041989581 MENKTSLGNNIYYNPFKPQDKSYFAGYLNAAMENIDSVFRELGKRLK cynodegmiGKEYTSENFFDAIFKENISLVEYERYVKLLSDYFPMARLLDKKEVPIKERKENFKKNFRGIIKAVRDLRNFYTHKEHGEVEITDEIFGVLDEMLKSTVLTVKKKKIKTDKTKEILKKSIEKQLDILCQKKLEYLKDTARKIEEKRRNQRERGEKKLVPRFEYSDRRDDLIAAIYNDAFDVYIDKKKDSLKESSKTKYNTESYPQQEEGDLKIPISKNGVVFLLSLFLSKQEVHAFKSKIAGFKATVIDEATVSHRKNSICFMATHEIFSHLAYKKLKRKVRTAEINYSEAENAEQLSIYAKETLMMQMLDELSKVPDVVYQNLSEDVQKTFIEDWNEYLKENNGDVGTMEEEQVIHPVIRKRYEDKFNYFAIRFLDEFAQFPTLRFQVHLGNYLHDSRPKEHLISDRRIKEKITVFGRLSELEHKKALFIKNTETNEDRKHYWEVFPNPNYDFPKENISVNDKDFPIAGSILDREKQPTAGKIGIKVNLLNQKYISEVDKAVKAHQLKQRNNKPSIQNIIEEIVPINGSNPKEIIVFGGQPTAYLSMNDIHSILYEFFDKWEKKKEKLEKKGEKELRKEIGKELEEKIVGKIQTQIQQIIDKDINAKILKPYQDDDSTAIDKEKLIKDLKQEQKILQKLKNEQTAREKEYQECIAYQEESRKIKRSDKSRQKYLRNQLKRKYPEVPTRKEILYYQEKGKVAVWLANDIKRFMPTDFKNEWKGEQHSLLQKSLAYYEQCKEELKNLLPQQKVFKHLPFELGGHFQQKYLYQFYTRYLDKRLEHISGLVQQAENFKNENKVFKKVENECFKFLKKQNYTHKGLDAQAQSVLGYPIFLERGFMDEKPTIIKGKTFKGNESLFTDWFRYYKEYQNFQTFYDTENYPLVELEKKQADRKRETKIYQQKKNDVFTLLMAKHIFKSVFKQDSIDRFSLEDLYQSREERLENQEKAKQTGERNTNYIWNKTVDLNLCDGKVTVENVKLKNVGNFIKYEYDQRVQTFLKYEENIKWQAFLIKESKEEENYPYIVEREIEQYEKVRREELLKEVHLIEEYILEKVKDKEILKKGDNQNFKYYILNGLLKQLKNEDVESYKVFNLNTKPEDVNINQLKQEATDLEQKAFVLTYIRNKFAHNQLPKKEFWDYCQEKYGKIEKEKTYAEYFAEVFKREKEALMK (SEQ ID NO: 161) Prevotella WP_042518169MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENN sp. P5-119ENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLIDGCEFLTSTEQPLSGMISKYYTVALRNTKERYGYKTEDLAFIQDNIKKITKDAYGKRKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKLPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSTDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEAETMRKQENGTFGNSGIRIRDFENVKRDDANPANYPYIVDTYTHYILENNKVEMFISDKGSSAPLLPLIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFQAMQKEEVTAENIASFGIAESDLPQKILDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFARSIPANAVDFYERYLIERKFYLTGLCNEIKRGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLNDDFQTFYQWKRNYHYMDMLKGEYDRKGSLQHCFTSVEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNCRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDIVSKEDIMEEFNKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGIVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 162) PrevotellaWP_044072147 MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENN sp. P4-76ENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDQASHYKTYDEKLIDGCEFLTSTEQPLSGMINNYYTVALRNMNERYGYKTEDLAFIQDKRFKFVKDAYGKKKSQVNTGFFLSLQDYNGDTQKKLHLSGVGIALLICLFLDKQYINIFLSRLPIFSSYNAQSEERRIIIRSFGINSIKQPKDRIHSEKSNKSVAMDMLNEIKRCPNELFETLSAEKQSRFRIISNDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEVETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVDTYTHYILENNKVEMFISDEETPAPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSKKTEKLIVDVHNRYKRLFKAMQKEEVTAENIASFGIAESDLPQKIIDLISGNAHGKDVDAFIRLTVDDMLADTERRTKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYNSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFVRSIPANAVDFYERYLIERKFYLIGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYDEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLNDDFQTFYQWKRNYRYMDMLRGEYDRKGSLQSCFTSVEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNSRNEYQKSEKVIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKKYTITSEGMKLKNYGDFFVLASDKRIGNLLELVGSDTVSKEDIMEEFKKYDQCRPEISSIVFNLEKWAFDTYPELSARVDREEKVDFKSILKILLNNKNINKEQSDILRKIRNAFDHNNYPDKGVVEIRALPEIAMSIKKAFGEYAIMK (SEQ ID NO: 163) PrevotellaWP_044074780 MNIPALVENQKKYFGTYSVMAMLNAQTVLDHIQKVADIEGEQNENN sp. P5-60ENLWFHPVMSHLYNAKNGYDKQPEKTMFIIERLQSYFPFLKIMAENQREYSNGKYKQNRVEVNSNDIFEVLKRAFGVLKMYRDLTNHYKTYEEKLIDGCEFLTSTEQPFSGMISKYYTVALRNTKERYGYKAEDLAFIQDNRYKFTKDAYGKRKSQVNTGSFLSLQDYNGDTTKKLHLSGVGIALLICLFLDKQYINLFLSRLPIFSSYNAQSEERRIIIRSFGINSIKQPKDRIHSEKSNKSVAMDMLNEVKRCPDELFTTLSAEKQSRFRIISDDHNEVLMKRSSDRFVPLLLQYIDYGKLFDHIRFHVNMGKLRYLLKADKTCIDGQTRVRVIEQPLNGFGRLEEVETMRKQENGTFGNSGIRIRDFENMKRDDANPANYPYIVETYTHYILENNKVEMFISDEENPTPLLPVIEDDRYVVKTIPSCRMSTLEIPAMAFHMFLFGSEKTEKLIIDVHDRYKRLFQAMQKEEVTAENIASFGIAESDLPQKIMDLISGNAHGKDVDAFIRLTVDDMLTDTERRIKRFKDDRKSIRSADNKMGKRGFKQISTGKLADFLAKDIVLFQPSVNDGENKITGLNYRIMQSAIAVYDSGDDYEAKQQFKLMFEKARLIGKGTTEPHPFLYKVFVRSIPANAVDFYERYLIERKFYLIGLSNEIKKGNRVDVPFIRRDQNKWKTPAMKTLGRIYSEDLPVELPRQMFDNEIKSHLKSLPQMEGIDFNNANVTYLIAEYMKRVLNDDFQTFYQWKRNYRYMDMLRGEYDRKGSLQHCFTSIEEREGLWKERASRTERYRKLASNKIRSNRQMRNASSEEIETILDKRLSNCRNEYQKSEKIIRRYRVQDALLFLLAKKTLTELADFDGERFKLKEIMPDAEKGILSEIMPMSFTFEKGGKIYTITSGGMKLKNYGDFFVLASDKRIGNLLELVGSNTVSKEDIMEEFKKYDQCRPEISSIVFNLEKWAFDTYPELPARVDRKEKVDFWSILDVLSNNKDINNEQSYILRKIRNAFDHNNYPDKGIVEIKALPEIAMSIKKAFGEYAIMK (SEQ ID  NO: 164)Phaeodactylibacter WP_044218239MTNTPKRRTLHRHPSYFGAFLNIARHNAFMIMEHLSTKYDMEDKNTL xiamenensisDEAQLPNAKLFGCLKKRYGKPDVTEGVSRDLRRYFPFLNYPLFLHLEKQQNAEQAATYDINPEDIEFTLKGFFRLLNQMRNNYSHYISNTDYGKFDKLPVQDIYEAAIFRLLDRGKHTKRFDVFESKHTRHLESNNSEYRPRSLANSPDHENTVAFVTCLFLERKYAFPFLSRLDCFRSTNDAAEGDPLIRKASHECYTMFCCRLPQPKLESSDILLDMVNELGRCPSALYNLLSEEDQARFHIKREEITGFEEDPDEELEQEIVLKRHSDRFPYFALRYFDDTEAFQTLRFDVYLGRWRTKPVYKKRIYGQERDRVLTQSIRTFTRLSRLLPIYENVKHDAVRQNEEDGKLVNPDVTSQFHKSWIQIESDDRAFLSDRIEHFSPHYNFGDQVIGLKFINPDRYAAIQNVFPKLPGEEKKDKDAKLVNETADAIISTHEIRSLFLYHYLSKKPISAGDERRFIQVDTETFIKQYIDTIKLFFEDIKSGELQPIADPPNYQKNEPLPYVRGDKEKTQEERAQYRERQKEIKERRKELNTLLQNRYGLSIQYIPSRLREYLLGYKKVPYEKLALQKLRAQRKEVKKRIKDIEKMRTPRVGEQATWLAEDIVFLTPPKMHTPERKTTKHPQKLNNDQFRIMQSSLAYFSVNKKAIKKFFQKETGIGLSNRETSHPFLYRIDVGRCRGILDFYTGYLKYKMDWLDDAIKKVDNRKHGKKEAKKYEKYLPSSIQHKTPLELDYTRLPVYLPRGLFKKAIVKALAAHADFQVEPEEDNVIFCLDQLLDGDTQDFYNWQRYYRSALTEKETDNQLVLAHPYAEQILGTIKTLEGKQKNNKLGNKAKQKIKDELIDLKRAKRRLLDREQYLRAVQAEDRALWLMIQERQKQKAEHEEIAFDQLDLKNITKILTESIDARLRIPDTKVDITDKLPLRRYGDLRRVAKDRRLVNLASYYHVAGLSEIPYDLVKKELEEYDRRRVAFFEHVYQFEKEVYDRYAAELRNENPKGESTYFSHWEYVAVAVKHSADTHFNELFKEKVMQLRNKFHHNEFPYFDWLLPEVEKASAALYADRVFDVAEGYYQKMRKLMRQ (SEQ ID NO: 165) FlavobacteriumWP_045968377 MDNNITVEKTELGLGITYNHDKVEDKHYFGGFFNLAQNNIDLVAQEF sp. 316KKRLLIQGKDSINIFANYFSDQCSITNLERGIKILAEYFPVVSYIDLDEKNKSKSIREHLILLLETINNLRNYYTHYYHKKIIIDGSLFPLLDTILLKVVLEIKKKKLKEDKTKQLLKKGLEKEMTILFNLMKAEQKEKKIKGWNIDENIKGAVLNRAFSHLLYNDELSDYRKSKYNTEDETLKDTLTESGILFLLSFFLNKKEQEQLKANIKGYKGKIASIPDEEITLKNNSLRNMATHWTYSHLTYKGLKHRIKTDHEKETLLVNMVDYLSKVPHEIYQNLSEQNKSLFLEDINEYMRDNEENHDSSEASRVIHPVIRKRYENKFAYFAIRFLDEFAEFPTLRFMVNVGNYIHDNRKKDIGGTSLITNRTIKQQINVFGNLTEIHKKKNDYFEKEENKEKTLEWELFPNPSYHFQKENIPIFIDLEKSKETNDLAKEYAKEKKKIFGSSRKKQQNTAKKNRETIINLVFDKYKTSDRKTVTFEQPTALLSFNELNSFLYAFLVENKTGKELEKIIIEKIANQYQILKNCSSTVDKTNDNIPKSIKKIVNTTTDSFYFEGKKIDIEKLEKDITIEIEKTNEKLETIKENEESAQNYKRNERNTQKRKLYRKYVFFTNEIGIEATWITNDILRFLDNKENWKGYQHSELQKFISQYDNYKKEALGLLESEWNLESDAFFGQNLKRMFQSNSTFETFYKKYLDNRKNTLETYLSAIENLKTMTDVRPKVLKKKWTELFRFFDKKIYLLSTIETKINELITKPINLSRGIFEEKPTFINGKNPNKENNQHLFANWFIYAKKQTILQDFYNLPLEQPKAITNLKKHKYKLERSINNLKIEDIYIKQMVDFLYQKLFEQSFIGSLQDLYTSKEKREIEKGKAKNEQTPDESFIWKKQVEINTHNGRIIAKTKIKDIGKFKNLLTDNKIAHLISYDDRIWDFSLNNDGDITKKLYSINTELESYETIRREKLLKQIQQFEQFLLEQETEYSAERKHPEKFEKDCNPNFKKYIIEGVLNKIIPNHEIEEIEILKSKEDVFKINFSDILILNNDNIKKGYLLIMIRNKFAHNQLIDKNLFNFSLQLYSKNENENFSEYLNKVCQNIIQEFKEKLK (SEQ ID NO: 166) PorphyromonasWP_046201018 MTEQSERPYNGTYYTLEDKHFWAAFLNLARHNAYITLTHIDRQLAYS gulaeKADITNDQDVLSFKALWKNFDNDLERKSRLRSLILKHFSFLEGAAYGKKLFESKSSGNKSSKNKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHSESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRYGHNDNPSFKHHFVDSEGMVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKPLYDRLREKDRARFRVPVDILPDEDDTDGGGEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKMIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYISQTTPHYHIEKGKIGLRFMPEGQHLWPSPEVGTTRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAIYDAFARDEINTLKELDACLADKGIRRGHLPKQMIAILSQEHKDMEEKIRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKKRLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLRARKAFLERIGRSDRMENRPFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYREVGFMAKAVPLYFERACEDRVQPFYDSPFNVGNSLKPKKGRFLSKEERAEEWERGKERFRDLEAWSHSAARRIEDAFAGIEYASPGNKKKIEQLLRDLSLWEAFESKLKVRADKINLAKLKKEILEAQEHPYHDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRPNVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEEAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCVFELTLRLEESLLTRYPHLPDESFRKMLESWSDPLLAKWPELHGKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKETVERIIQV (SEQ ID NO: 167)WP_047431796 Chryseo METQTIGHGIAYDHSKIQDKHFFGGFLNLAENNIKAVLKAFSEKFNVGbacterium NVDVKQFADVSLKDNLPDNDFQKRVSFLKMYFPVVDFINIPNNRAKF sp.RSDLTTLFKSVDQLRNFYTHYYHKPLDFDASLFILLDDIFARTAKEVR YR477QKMKDDKTRQLLSKSLSEELQKGYELQLERLKELNRLGKKVNIHDQLGIKNGVLNNAFNHLIYKDGESFKTKLTYSSALTSFESAENGIEISQSGLLFLLSMFLKRKEIEDLKNRNKGFKAKVVIDEDGKVNGLKFMATHWVFSYLCFKGLKSKLSTEFHEETLLIQIIDELSKVPDELYCAFDKETRDKFIEDINEYVKEGHQDFSLEDAKVIHPVIRKRYENKFNYFAIRFLDEFVKFPSLRFQVHVGNYVHDRRIKNIDGTTFETERVVKDRIKVFGRLSEISSYKAQYLSSVSDKHDETGWEIFPNPSYVFINNNIPIHISVDTSFKKEIADFKKLRRAQVPDELKIRGAEKKRKFEITQMIGSKSVLNQEEPIALLSLNEIPALLYEILINGKEPAEIERIIKDKLNERQDVIKNYNPENWLPASQISRRLRSNKGERIINTDKLLQLVTKELLVTEQKLKIISDNREALKQKKEGKYIRKFIFTNSELGREAIWLADDIKRFMPADVRKEWKGYQHSQLQQSLAFYNSRPKEALAILESSWNLKDEKIIWNEWILKSFTQNKFFDAFYNEYLKGRKKYFAFLSEHIVQYTSNAKNLQKFIKQQMPKDLFEKRHYIIEDLQTEKNKILSKPFIFPRGIFDKKPTFIKGVKVEDSPESFANWYQYGYQKDHQFQKFYDWKRDYSDVFLEHLGKPFINNGDRRTLGMEELKERIIIKQDLKIKKIKIQDLFLRLIAENLFQKVFKYSAKLPLSDFYLTQEERMEKENMAALQNVREEGDKSPNIIKDNFIWSKMIPYKKGQIIENAVKLKDIGKLNVLSLDDKVQTXLSYDDAKPWSKIALENEFSIGENSYEVIRREKLFKEIQQFESEILFRSGWDGINHPAQLEDNRNPKFKMYIVNGILRKSAGLYSQGEDIWFEYNADFNNLDADVLETKSELVQLAFLVTAIRNKFAHNQLPAKEFYFYIRAKYGFADEPSVALVYLNFTKYAINEFKKVMI (SEQ ID NO: 168) Riemerella WP_049354263MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHL anatipestiferNRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQREKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEENKKHFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSNQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEIKDGANRIAKYPYNSGFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIKSSKANSTEFWFIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSEDLMLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKREEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMYLPVKVYPATAFGEVQYHKTPIRTVYIREEHTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHTSLSSLENEFRALLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYKEALHAPIPLFTVAQPTTEEKDGLGIAEALLKVLREYCEIVKSQI (SEQ ID NO: 169)Porphyromonas WP_052912312MTEQNEKPYNGTYYTLEDKHFWAAFFNLARHNAYITLAHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDKYGNNDNPFFKHHFVDREEKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKLLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQLLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEASAEKVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFVGIEYASWENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYHDFKSWQKFERELRLYKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEOGSLNVLNHVKPMRLPVVVYRADSRGHVHKEEAPLAIYYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPFILPDESFREMLESWSDPLLDKWPDLQREVRLLIAVRNAFSHNQYPMYDETIFSSIRKYDPSSLDAIEERMGLNIAHRLSEEVKLAKEMVERIIQA (SEQ ID  NO: 170) PorphyromonasWP_058019250 MTEQNEKPYNGTYYTLKDKHFWAAFFNLARHNAYITLTHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFESQSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPMFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREGKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTETYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRACFRVPVDILSDEDDTDGAEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDCFETGDKPYTTQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRFTAEAFLSVHELMPMMFYYFLLREKYSEEVSAERVQGRIKRVIEDVYAVYDAFARDEINTRDELDACLADKGIRRGHLPRQMIAILSQKHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDRVENHRFLLLKEPKTDRQTLVAGWKGEFHLPRGIFTEAVRDCLIEMGLDEVGSYKEVGFMAKAVPLYFERACKDRVOPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRDLEAWSHSAARRIEDAFAGIENASRENKKKIEQLLQDLSLWETFESKLKVKADKINIAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIITWMMCRDLMEENKVEGLDTGTLYLKDIRTDVQEQGSLNVLNHVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGALAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRYPHLPDENFRKMLESWSDPLLDKWPDLHRKVRLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAIEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 171)Flavobacterium WP_060381855MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKEFKTR columnareINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKVYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKFRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGLVFLISFFLHRKEFQVFTSGLEGFKAKVNTIKEEEISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLGLVTEYKKNVYLKETSNIDLSRFPLFPSPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNRKRNLTFEKYNKEQSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLDSPQKDNIPTTLTKTISTDTSVTFENQPIDIPRLKNALQKELTLTQEKLLNVKQHEIEVDNYNRNKNTYKFKNQPKDKVDDNKLQRKYVFYRNEIGQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKDLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGFIGLPPKILKKELSKRLNYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDKIENDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKTDREKTKADAKAYQKRNDSFLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELOEYEKVRSKKLLKQVQELEKQILDKFYDFSNNATHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILIKYYDYNTEKLKEKIKNMDEDEKAKIVNTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFRYDLATRFVPKKEEEYFACYFNRVFETITTELWENKKKAKEIV (SEQ ID NO: 172) Porphyromonas WP_061156470MTEQNERPYNGTYYTLEDKHFWAAFFNLARHNAYITLTHIDRQLAYS gingivalisKADITNDEDILFFKGQWKNLDNDLERKARLRSLILKHFSFLEGAAYGKKLFENKSSGNKSSKKKELTKKEKEELQANALSLDNLKSILFDFLQKLKDFRNYYSHYRHPESSELPLFDGNMLQRLYNVFDVSVQRVKRDHEHNDKVDPHRHFNHLVRKGKKDRCGNNDNPFFKHHFVDREGKVTEAGLLFFVSLFLEKRDAIWMQKKIRGFKGGTEAYQQMTNEVFCRSRISLPKLKLESLRTDDWMLLDMLNELVRCPKSLYDRLREEDRARFRVPVDILSDEDDTDGTEEDPFKNTLVRHQDRFPYFALRYFDLKKVFTSLRFHIDLGTYHFAIYKKNIGEQPEDRHLTRNLYGFGRIQDFAEEHRPEEWKRLVRDLDYFETGDKPYITQTTPHYHIEKGKIGLRFVPEGQHLWPSPEVGATRTGRSKYAQDKRLTAEAFLSVHELMPMMFYYFLLREKYSEEVSAEKVQGRIKRVIEDVYAVYDAFARGEIDTLDRLDACLADKGIRRGHLPRQMIAILSQEHKDMEEKVRKKLQEMIADTDHRLDMLDRQTDRKIRIGRKNAGLPKSGVIADWLVRDMMRFQPVAKDTSGKPLNNSKANSTEYRMLQRALALFGGEKERLTPYFRQMNLTGGNNPHPFLHETRWESHTNILSFYRSYLKARKAFLQSIGRSDREENHRFLLLKEPKTDRQTLVAGWKSEFHLPRGIFTEAVRDCLIEMGYDEVGSYKEVGFMAKAVPLYFERACKDRVQPFYDYPFNVGNSLKPKKGRFLSKEKRAEEWESGKERFRLAKLKKEILEAKEHPYLDFKSWQKFERELRLVKNQDIRRWMMCRDLMEENKVEGLDTGTLYLKDIRTEVQEQGSLNVLNRVKPMRLPVVVYRADSRGHVHKEQAPLATVYIEERDTKLLKQGNFKSFVKDRRLNGLFSFVDTGGLAMEQYPISKLRVEYELAKYQTARVCAFEQTLELEESLLTRCPHLPDKNFRKMLESWSDPLLDKWPDLQREVWLLIAVRNAFSHNQYPMYDEAVFSSIRKYDPSSPDAEERMGLNIAHRLSEEVKQAKEMAERIIQA (SEQ ID NO: 173) PorphyromonasWP_061156637 MNTVPASENKGQSRTVEDDPQYFGLYLNLARENLIEVESHVRIKFGK gingivalisKKLNEESLKQSLLCDHLLSVDRWTKVYGHSRRYLPFLHYFDPDSQIEKDHDSKTGVDPDSAQRLIRELYSLLDFLRNDFSHNRLDGTTFEHLEVSPDISSFITGTYSLACGRAQSRFADFFKPDDFVLAKNRKEQLISVADGKECLTVSGLAFFICLFLDREQASGMLSRIRGFKRTDENWARAVHETFCDLCIRHPHDRLESSNTKEALLLDMLNELNRCPRILYDMLPEEERAQFLPALDENSMNNLSENSLNEESRLLWDGSSDWAEALTKRIRHQDRFPYLMLRFIEEMDLLKGIRFRVDLGEIELDSYSKKVGRNGEYDRTITDHALAFGKLSDFQNEEEVSRMTSGEASYPVRFSLFAPRYTAIYDNKIGYCHTSDPVYPKSKTGEKRALSNPQSMGFISVHDLRKLLLMELLCEGSFSRMQSGFLRKANRILDETAFGKLQFSALFPEMRHRFIPPQNPKSKDRREKAETTLEKYKQEIKGRKDKLNSQLLSAFDMNQRQLPSRLLDEWMNIRPASHSVKLRTYVKQLNEDCRLRLRKFRKDGDGKARAIPLVGEMATFLSQDIVRMIISEETKKLITSAYYNEMQRSLAQYAGEENRRQFRAIVAELHLLDPSSGHPFLSATMETAHRYTEDFYKCYLEKKREWLAKTFYRPEQDENTKRRISVFFVPDGEARKLLPLLIRRRMKEQNDLQDWIRNKQAHPIDLPSHLFDSKIMELLKVKDGKKKWNEAFKDWWSTKYPDGMQPFYGLRRELNIHGKSVSYIPSDGKKFADCYTHLMEKTVQDKKRELRTAGKPVPPDLAADIKRSFHRAVNEREFMLRLVQEDDRLMLMAINKMMTDREEDILPGLKNIDSILDKENQFSLAVHAKVLEKEGEGGDNSLSLVPATIEIKSKRKDWSKYIRYRYDRRVPGLMSHFPEHKATLDEVKTLLGEYDRCRIKIFDWAFALEGAIMSDRDLKPYLHESSSREGKSGEHSTLVKMLVEKKGCLTPDESQYLILIRNKAAHNQFPCAAEMPLIYRDVSAKVGSIEGSSAKDLPEGSSLVDSLWKKYEMIIRKILPILDPENRFFGKLLNNMSQPINDL (SEQ ID NO: 174) RiemerellaWP_061710138 MFFSFHNAQRVIFKHLYKAFDASLRMVKEDYKAHFTVNLTRDFAHLanatipestifer NRKGKNKQDNPDFNRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQSEKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRCPKLLYEKLSEKDKKCFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGYTEQEKRHLTRTLLNFGRLQDFTEINRPQEWKALTKDLDYNETSNQPFISKTTPHYITDNKIGFRLRTSKELYPSLEVKDGANRIAKYPYNSDFVAHAFISISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVVYDAQNQPIKSSKANSTESRLIRRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKHQPWEPYQYCLLLKVPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKHGRVGFISRAITLYFKEKYQDKHQSFYNLSYKLEAKAPLLKKEEHYEYWQQNKPQSPTESQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDIMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPTTAFGEVQYHETPIRTVYIREEQTKALKMGNFKALVKDRHLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLSLEEKLLNKHASLSSLENEFRTLLEEWKKKYAASSMVTDKHLAFIASVRNAFCFLNQYPFYKETLFIAPILLFTVAQPTTEEKDGLGIAEALLRVLREYCEIVKSQI (SEQ ID NO: 175)Flavobacterium WP_063744070MSSKNESYNKQKTFNHYKQEDKYFFGGFLNNADDNLRQVGKEFKTR columnareINFNHNNNELASVFKDYFNKEKSVAKREHALNLLSNYFPVLERIQKHTNHNFEQTREIFELLLDTIKKLRDYYTHHYHKPITINPKIYDFLDDTLLDVLITIKKKKVKNDTSRELLKEKLRPELTQLKNQKREELIKKGKKLLEENLENAVFNHCLRPFLEENKTDDKQNKTVSLRKYRKSKPNEETSITLTQSGILVFLMSFFLHRKEFQVFTSGLEGFKAKVNTIKEEKISLNKNNIVYMITHWSYSYYNFKGLKHRIKTDQGVSTLEQNNTTHSLTNTNTKEALLTQIVDYLSKVPNEIYETLSEKQQKEFEEDINEYMRENPENEDSTFSSIVSHKVIRKRYENKFNYFAMRFLDEYAELPTLRFMVNFGDYIKDRQKKILESIQFDSERIIKKEIHLFEKLGLVTEYKKNVYLKETSNIDLSRFPLFPSPSYVMANNNIPFYIDSRSNNLDEYLNQKKKAQSQNRKRNLTFEKYNKEOSKDAIIAMLQKEIGVKDLQQRSTIGLLSCNELPSMLYEVIVKDIKGAELENKIAQKIREQYQSIRDFTLNSPQKDNIPTTLIKTISTDTSVTFENQPIDIPRLKNAIQKELALTQEKLLNVKQHEIEVNNYNRNKNTYKFKNQPKDKVDDNKLQRKYVFYRNEIGQEANWLASDLIHFMKNKSLWKGYMHNELQSFLAFFEDKKNDCIALLETVFNLKEDCILTKDLKNLFLKHGNFIDFYKEYLKLKEDFLNTESTFLENGFIGLPPKILKKELSKRLNYIFIVFQKRQFIIKELEEKKNNLYADAINLSRGIFDEKPTMIPFKKPNPDEFASWFVASYQYNNYQSFYELTPDKIENDKKKKYKNLRAINKVKIQDYYLKLMVDTLYQDLFNQPLDKSLSDFYVSKTDREKIKADAKAYQKRNDSFLWNKVIHLSLQNNRITANPKLKDIGKYKRALQDEKIATLLTYDDRTWTYALQKPEKENENDYKELHYTALNMELQEYEKVRSKKLLKQVQELEKQILDKFYDFSNNATHPEDLEIEDKKGKRHPNFKLYITKALLKNESEIINLENIDIEILIKYYDYNTEKLKEKIKNMDEDEKAKIVNTKENYNKITNVLIKKALVLIIIRNKMAHNQYPPKFIYDLATRFVPKKEEEYFACYFNRVFETITTELWENKKKAKEIV (SEQ ID NO: 176) Riemerella WP_064970887MEKPLPPNVYTLKHKFFWGAFLNIARHNAFITICHINEQLGLTTPPNDD anatipestiferKIADVVCGTWNNILNNDHDLLKKSQLTELILKHFPFLAAMCYHPPKKEGKKKGSQKEQQKEKENEAQSQAEALNPSELIKVLKTLVKQLRTLRNYYSHHSHKKPDAEKDIFKHLYKAFDASLRMVKEDYKAHFTVNLTQDFAHLNRKGKNKQDNPDFDRYRFEKDGFFTESGLLFFTNLFLDKRDAYWMLKKVSGFKASHKQSEKMTTEVFCRSRILLPKLRLESRYDHNQMLLDMLSELSRYPKLLYEKLSEEDKKRFQVEADGFLDEIEEEQNPFKDTLIRHQDRFPYFALRYLDLNESFKSIRFQVDLGTYHYCIYDKKIGDEQEKRHLTRTLLSFGRLQDFTEINRPQEWKALTKDLDYKETSKQPFISKTTPHYHITDNKIGFRLGTSKELYPSLEVKDGANRIAQYPYNSDFVAHAFISVHELLPLMFYQHLTGKSEDLLKETVRHIQRIYKDFEEERINTIEDLEKANQGRLPLGAFPKQMLGLLQNKQPDLSEKAKIKIEKLIAETKLLSHRLNTKLKSSPKLGKRREKLIKTGVLADWLVKDFMRFQPVAYDAQNQPIESSKANSTEFQLIQRALALYGGEKNRLEGYFKQTNLIGNTNPHPFLNKFNWKACRNLVDFYQQYLEQREKFLEAIKNQPWEPYQYCLLLKIPKENRKNLVKGWEQGGISLPRGLFTEAIRETLSKDLTLSKPIRKEIKKHGRVGFISRAITLYFREKYQDDHQSFYDLPYKLEKASPLPKKEHYEYWQQNKPQSPTELQRLELHTSDRWKDYLLYKRWQHLEKKLRLYRNQDVMLWLMTLELTKNHFKELNLNYHQLKLENLAVNVQEADAKLNPLNQTLPMVLPVKVYPATAFGEVQYQETPIRTVYIREEQTKALKMGNFKALVKDRRLNGLFSFIKEENDTQKHPISQLRLRRELEIYQSLRVDAFKETLNLEEKLLKKHTSLSSVENKFRILLEEWKKEYAASSMVTDEHIAFIASVRNAFCHNQYPFYEEALHAPIPLFTVAQQTTEEKDGLGIAEALLRVLREYCEIVKSQI (SEQ ID NO: 177)Sinomicrobium WP_072319476.1MESTTTLGLHLKYQHDLFEDKHYTGGGVNLAVQNIESIFQAFAERYGT oceaniQNPLRKNGVPAINNIFHDNISISNYKEYLKFLKQYLPVVGFLEKSNEINIFEFREDFEILINAIYKLRHFYTHYYHSPIKLEDRFYTCLNELFVAVAIQVKKHKMKSDKTRQLLNKNLHQLLQQLIEQKREKLKDKKAEGEKVSLDTKSIENAVLNDAFVFHLLDKDENIRLNYSSRLSEDIITKNGITLSISGLLFLLSLFLQRKEAEDLRSRIEGFKGKGNELRFMATHWVFSYLNVKRIKHRLNTDFQKETLLIQIADELSKVPDEVYKTLDHENRSKFLEDINEYIREGNEDASLNESTVVHGVIRKRYENKFHYLVLRYLDEFVDFPSLRFQVHLGNYIHDRRDKVIDGTNFITNRVIKEPIKVFGKLSHVSKLKSDYMESLSREHKNGWDVFPNPSYNFVGHNIPIFINLRSASSKGKELYRDLMKIKSEKKKKSREEGIPMERRDGKPTKIEISNQIDRNIKDNNFKDIYPGEPLAMLSLNELPALLFELLRRPSITPQDIEDRMVEKLYERFQIIRDYKPGDGLSTSKISKKLRKADNSTRLDGKKLLRAIQTETRNAREKLHTLEENKALQKNRKRRTVYTTREQGREASWLAQDLKRFMPIASRKEWRGYHHSQLQQILAFYDQNPKQPLELLEQFWDLKEDTYVWNSWIHKSLSQHNGFVPMYEGYLKGRLGYYKKLESDIIGFLEEHKVLKRYYTQQHLNVIFRERLYFIKTETKQKLELLARPLVFPRGIFDDKPTFVQDKKVVDHPELFADWYVYSYKDDHSFQEFYHYKRDYNEIFETELSWDIDFKDNKRQLNPSEQMDLFRMKWDLKIKKIKIQDIFLKIVAEDIYLKIFGHKIPLSLSDFYISRQERLTLDEQAVAQSMRLPGDTSENQIKESNLWQTTVPYEKEQIREPKIKLKDIGKFKYFLQQQKVLNLLKYDPQHVWTKAELEEELYIGKHSYEVVRREMLLQKCHQLEKHILEQFRFDGSNHPRELEQGNHPNFKMYIVNGILTKRGELEIEAENWWLELGNSKNSLDKVEVELLTMKTIPEQKAFLLILIRNKFAHNQLPADNYFHYASNLMNLKKSDTYSLFWFTVADTIVQEFMSL (SEQ ID NO: 178)Reichenbachiella WP_073124441.1MKTNPLIASSGEKPNYKKFNTESDKSFKKIFQNKGSIAPIAEKACKNFE agariperforansIKSKSPVNRDGRLHYFSVGHAFKNIDSKNVFRYELDESQMDMKPTQFLALQKEFFDFQGALNGLLKHIRNVNSHYVHTFEKLEIQSINQKLITFLIEAFELAVIHSYLNEEELSYEAYKDDPQSGQKLVQFLCDKFYPNKEHEVEERKTILAKNKRQALEHLLFIEVTSDIDWKLFEKHKVFTISNGKYLSFHACLFLLSLFLYKSEANQLISKIKGFKRNDDNQYRSKRQIFTFFSKKFTSQDVNSEEQFILVKFRDVIQYLNHYPSAWNKHLELKSGYPQMTDKLMRYIVEAEIYRSFPDQTDNHRFLLFAIREFFGQSCLDTWTGNTPINFSNQEQKGFSYEINTSAEIKDIETKLKALVLKGPLNFKEKKEQNRLEKDLRREKKEQPTNRVKEKLLTRIQHNMLYVSYGRNQDRFMDFAARFLAETDYFGKDAKFKMYQFYTSDEQRDHLKEQKKELPKKEFEKLKYHQSKLVDYFTYAEQQARYPDWDTPFVVENNAIQIKVTLFNGAKKIVSVQRNLMLYLLEDALYSEKRENAGKGLISGYFVHHQKELKDQLDILEKETEISREQKREFKKLLPKRLLHRYSPAQINDTTEWNPMEVILEEAKAQEORYQLLLEKAILHQTEEDFLKRNKGKQFKLRFVRKAWHLMYLKELYMNKVAEHGHHKSFHITKEEFNDFCRWMFAFDEVPKYKEYLCDYFSQKGFFNNAEFKDLIESSTSLNDLYEKTKQRFEGWSKDLTKQSDENKYLLANYESMLKDDMLYVNISHFISYLESKGKINRNAHGHIAYKALNNVPFSLIEEYYYKDRLAPEEYKSHGKLYNKLKTVKLEDALLYEMAMHYLSLEPALVPKVKTKVKDILSSNIAFDIKDAAGHHLYHLLIPFHKIDSFVALINHQSQQEKDPDKTSFLAKIQPYLEKVKNSKDLKAVYHYYKDTPHTLRYEDLNMIHSHIVSQSVQFTKVALKLEEYFIAKKSITLQIARQISYSEIADLSNYFTDEVRNTAFHFDVPETAYSMILQGIESEFLDREIKPQKPKSLSELSTQQVSVCTAFLETLHNNLFDRKDDKKERLSKARERYFEQIN (SEQ ID NO: 179)

Example 28: Identification of C2c2 Orthologs

To improve or otherwise alter the properties of the C2c2 enzyme,modifications of amino acids are implemented. The changeable residuesare identified as a subset of the conserved charged residues. Theseresidues have >80% conservation in the alignment of FIG. 53 . These canbe changed to an uncharged residue (typically an alanine). One or moreof the indicated residue is mutated. Amino acid residue numberingcorresponds to the consensus numbering as indicated in FIG. 66 (topline), and reproduced below:

(SEQ ID NO: 180) MWISIKTLIHHLGVLFFCDMGNLFGHMKIXKVXHEKRXAKXKXPXKKVXVKRKYSGGGLLLNYNENPNKNKSXENILIKKKTSFXXLKSSSKLBKTINKPDXKKXXXXLQWFLSEIVKKINRRNGLVLSDMLSVDKRXXEKIXEKXXXLKYFXXXXXXLXKLHQEKPSKKLFNLKDLKEXEEXVLFLKXKFKNEJXYXXENDXXKDIEKILXEXLRXGFXPADKKLKXKFLIEXXWGIFSXXXKLEPYXIQEDFXEXYIEDFKKLNKXKXJXKSIENNKIVSQKSSDSQIYEXGKNIIMSXXGXIESIIEXXSKRKXXLDKYATXXLXEKLLLDEXLXIEQXXXNXXEXXDKLASNLKXYXLXKLYFYVKXDKKKSXXEVAKAAVSAAKDXNKDKYQNEWXXHEXRKEDKRDFIXXXLETXXIXKXIXKVKXXIXKXAXXEAXEXIKXXNIGKYRXXJDLFELEEDNXLNQFXXFVNIEXXKFFXHYXPNXIKRIXXXKNDAXAXXLKXGELXKKVEKQLKNGALSIYXIXXGKAVYYXXFAMKXLADSDYWTXKDLEXIKISEAFLRKFIGACSFAYXSLXAXNILQPECXXDILGKGDLLXKATVNIXQXXSEHIMYLGKLREINDIDXLLXFKEDIAKSTXKXGXGXLXKNLTQFFGGESTWDNKIFXAAYXXXLXGXXENEDFLGWALRGAIXSIRNEXFHSFKIKKHXXXXFLNIXNFIXXKLXEFEKXXXXKXKEXXHXXXTSYXXXLIKKLFXNEXXKXXLPXXIKELKLKSSGVXMYYSXDDLKKLLENIYFKFSLLKIXEENXEXAXFVPSFKKVYXRADGVKGFDYQXXXTRXHAYXLKLXPFFDXEEXEXEAFNARYYLLKXIYYNXILEXXXEENEXXXXFLPKFXXXNNXAFREXXNFXADXIEXYYKRLQINKKKGAXKXXKKKXQXKVXNXYNRKXFAYAFENIRXMXFXETPREYMQYIQSEYXIENNGKEXKKSXXENKRNKDXFXHXEKFLLQVFIKGFDXYJDXRXENFXFILXPEPQNGTKEYLYEEXXAILDEXXXXNXLRXXXITXNKXLKLXEFJPEXKSDIKVXPXLVEEIYDYIKKIKINKIKKDXEJAFWQDAALYLFCEKLLDARHLSXXLRXELIKYKQFXKDIKXRAXXNGNXINHSXXXNXXXVXECTDELEIIELXLLLNDRXSNDFKDYFDDEEAXIXXXXLCRIIFYAEYLXKYXKEEDDXXXXAEXXXFXALEPFCQSDTAREAKNDIYXDGGXNPELRVPILNRGIXQXKKIYGIEXXLEKLFDKNXLFBIDGXBIPXFKVSEEXAIIXEXXEKKXEIXEXSQYKXRGELHTEWXQKAREIEEYXXXXXKXKFXKKPQNXXFEKRFIEKHGQEYKKAXXXIXEYXWLKNKVEXNXLNELHELLIXLLGRLIGYSALFERDLQYFXNGFHYXCLNNDXEKLAXYXNJSXVXXKNRXIXKAXLYQIFAMYXXGLPFYSKDXDXXXAXXSGXKXSXXXXSXXTAGXGKKJKKFKKYSXYXLIXXXLXXDXSKKLDXYLAGLELFENXEEHDNXTEXIRNYIAHFNYLXXAGXXADXSLLELYNXLRDRLXSYDRKLKNAVSKSLIDILDRHGMILKFKFKXXXKLIGXNDXXXXAIKHKDXARITIXEPNGVTSEXFTYKLLXXVAALEIXSLEPKKIRHLXXXARLLYYPKXATAQSQPDQKXXXKXKKKNIXKGYIERXTNQVSSNQEEYCELVKKLLETXXLXXLAVXGVAXBIGLHISRLRRIREDAIIVGRRYRFRVEIYVPP KSNTSKLNAADLVRID

Mutated residues based on consensus sequence using MUSCLE alignment(www.ebi.ac.uk/Tools/msa/muscle/). Corresponding positions in Lshindicated

consensus Lsh consensus Lsh K28 R9 E839 K1134 K31 E12 R885 K1187 R44 R29E894 E1196 E162 E154 R895 R1197 E184 E179 D896 D1198 K262 R362 K942K1254 E288 K353 R960 (HEPN) R1278 K357 K429 H965 (HEPN) H1283 E360 Y432D990 S1310 K338 K405 K992 R1312 R441 (HEPN) K558 K994 N1314 H446 (HEPN)N563 E471 D616 K482 K628 K525 E679 K558 K711 D707 D943 R790 I1067 K811K1103 R833 K1128

C2c2 proteins having any one or more of the above amino acid residues,alone, or in combination, mutated display altered specificity and/oractivity and/or alternative PAM recognition.

Example 29

Based on the alignment of Lw2 and FSL (FIG. 67 ), the followingconserved residues were identified:

M35 K198 I478 A593 R717 F825 K36 N201 E479 L597 H722 Y829 T38 Y222 K494I601 F740 K831 K39 D253 R495 L602 F742 D837 I57 I266 N498 E611 K768 L852E65 F267 S501 E613 I774 F858 G66 S280 E519 D630 K778 E867 L68 I303 N524I631 I783 A871 N84 N306 Y529 G633 L787 L875 T86 R331 V530 K641 S789 K877E88 Y338 G534 N646 V792 Y880 I103 K389 K535 V669 Y796 Y881 N105 Y390Y539 F676 D799 F884 E123 K391 T549 S678 F812 F888 R128 I434 D551 N695N818 F896 R129 K435 R577 E703 P820 N901 K139 L458 E580 A707 F821 V903L152 D459 A581 I709 V822 N915 L194 E462 F582 I713 P823 K916 N196 L463I587 I716 S824 R918 Q920 I1075 K1243 K1341 K1466 A1550 V1684 E951 K1076Y1244 N1342 R1509 K1553 K1685 P956 F1092 G1245 K1343 N1510 S1554 E1689Y959 K1097 D1255 N1350 I1512 D1557 Q964 L1099 K1261 L1352 A1513 I1558I969 L1104 S1263 L1355 H1514 L1559 N994 L1107 L1267 L1356 N1516 G1563F1000 K1113 E1269 I1359 Y1517 F1568 I10001 Y1114 K1274 L1360 L1529 I1612Q1003 E1149 I1277 R1362 L1530 L1651 F10005 E1151 E1278 V1363 E1534 E1652K1007 I1153 L1289 G1364 L1536 K1655 G1008 L1155 H1290 Y1365 R1537 H1658F1009 L1158 A1294 I1369 Y1543 L1659 N1019 D1166 N1320 R1371 D1544 K1663L1020 L1203 K1325 D1372 R1545 T1673 K1021 D1222 E1327 F1385 K1546 S1677I1023 G1224 Y1328 E1391 L1547 E1678 N1028 I1228 I1334 D1459 K1548 E1679E1070 R1236 Y1337 K1463 N1549 C1681

One or more of the indicated residue is mutated. Amino acid residuenumbering corresponds to the numbering as indicated in FIG. 67A-67F(middle line between the two orthologous aligned sequences, indicatingidentical residues).

Any one or more of the residues indicated in FIG. 67A-67F, which areidentical between Lew2 C2c2 and Lib C2c2 are mutated for modifying theC2c2 protein activity, specificity, or functionality. C2c2 proteinshaving any one or more of the above amino acid residues, alone, or incombination, mutated display altered specificity and/or activity and/oralternative PAM recognition.

Example 30: Methods

Cloning of Orthologs for Activity Screen and Recombinant Expression

We synthesized human codon-optimized versions of fifteen Cas13aorthologs (Genscript, Jiangsu, China) (Supplementary Table 9) and clonedthem into a pACYC184 under expression by a pLac promoter. Adjacent tothe Cas13a expression cassette, we cloned the ortholog's correspondingdirect repeats flanking either a beta-lactamase targeting ornon-targeting spacer. Spacer array expression was driven by the J23119promoter.

For purification of LwaCas13a, we cloned the mammalian codon-optimizedLwaCas13a sequence into a bacterial expression vector for proteinpurification (6×His (SEQ ID NO: 577)/Twin Strep SUMO, a pET-basedexpression vector received as a gift from Ilya Finkelstein, Universityof Texas-Austin).

All plasmids used in this study are listed in Supplementary Table 1.

Bacterial In Vivo Testing for Cas13a Activity and PFS Identity

The screen functions as follows. Briefly, Cas13a is programmed to targeta 5′ stretch of sequence on the β-lactamase transcript flanked byrandomized PFS nucleotides. Cas13a cleavage activity results in death ofbacteria under ampicillin selection and PFS depletion is subsequentlyanalyzed by next generation sequencing. In order to allow forquantitative comparisons between orthologs, we cloned each Cas13aortholog under a pLac promoter along with a single-spacer CRISPR arraynearby under expression of the pJ23119 small RNA promoter.

To test for activity of Cas13a orthologs, 90 ng of ortholog expressionplasmids, with either targeting or non-targeting guide, wasco-transformed with 25 ng of a previously described beta-lactamasetarget plasmid⁵⁸ into NovaBlue Singles competent cells (Millipore).Post-transformation, cells were diluted, plated on LB-agar supplementedwith 100 μg/μL ampicillin and 25 μg/μL chloramphenicol, and incubated at37° C. overnight. Transformants were counted next day.

For determination of LshCas13a and LwaCas13a PFS identity, 40 ng ofortholog expression plasmids with either targeting or non-targetingspacer was co-transformed with 25 ng of beta-lactamase target plasmidinto 2 aliquots of NovaBlue GigaSingles (Millipore) per biologicalreplicate. Two biological replicates were performed.Post-transformation, cells were recovered at 37° C. in 500 μL of SOC(Thermo Fisher Scientific) per biological replicate for 1 hour, platedon bio-assay plates (Corning) with LB-agar (Affymetrix) supplementedwith 100 μg/μL ampicillin and 25 μg/μL chloramphenicol, and incubated at37° C. for 16 hours. Colonies were then harvested by scraping andplasmid DNA was purified with NucleoBond Xtra EF (Macherey-Nagel) forsubsequent sequencing.

Harvested plasmid samples were prepared for next generation sequencingby PCR with barcoding primers and Illumina flow cell handles usingNEBNext High Fidelity 2×Master Mix (New England Biosciences). PCRproducts were pooled and gel extracted using a Zymoclean gel extractionkit (Zymo Research) and sequenced using a MiSeq next generationsequencing machine (Illumina).

Computational Analysis of PFS

From next generation sequencing of the LshCas13a and LwaCas13a PFSscreening libraries, we aligned the sequences flanking the randomizedPFS region and extracted the PFS identities. We collapsed PFS identitiesto 4 nucleotides to improve sequence coverage, counted the frequency ofeach unique PFS, and normalized to total read count for each librarywith a pseudocount of 1. Enrichment of each distribution as displayed inFIG. 1E was calculated against the pACYC184 control (no protein/guidelocus) as −log₂(f_(condition)/f_(pACYC184)), where f_(condition) is thefrequency of PFS identities in the experimental condition andf_(pACYC184) is the frequency of PFS identities in the pACYC184 control.For analysis of a conserved PFS motif, top depleted PFS identities werecalculated using each condition's non-targeting control as follows: −log2(f_(i,targeting)/f_(i,non-targeting)) where f_(i,targeting) is thefrequency of PFS identities in condition i with targeting spacer andf_(i,non-targeting) is the frequency of PFS identities in condition iwith non-targeting spacer. PFS motifs were analyzed for a range ofthresholds as shown in Extended Data FIG. 1D, 1E.

Purification of LwaCas13a

Purification of LwaCas13a was performed as previously described⁶⁰.Briefly, LwaCas13a bacterial expression vectors were transformed intoRosetta 2(DE3)pLysS singles Competent Cells (Millipore) and 4 L ofTerrific Broth 4 growth media (TB) was seeded with a starter culture.Cell protein expression was induced with IPTG and after overnightgrowth, cell pellet was harvested and stored at −80° C. Following celllysis, protein was bound using a StrepTactin Sepharose resin (GE) andprotein was eluted by SUMO protease digestion (Thermo Fisher). Proteinwas further purified by cation exchange using a HiTrap SP HP cationexchange column (GE Healthcare Life Sciences) and subsequently by gelfiltration using a Superdex 200 Increase 10/300 GL column (GE HealthcareLife Sciences), both steps via FPLC (AKTA PURE, GE Healthcare LifeSciences). Final fractions containing LwaCas13a protein were pooled andconcentrated into Storage Buffer (600 mM NaCl, 50 mM Tris-HCl pH 7.5, 5%Glycerol, 2 mM DTT) and aliquots were frozen at −80° C. for long-termstorage.

Cloning of Mammalian Expression Constructs

The human codon optimized Cas13a gene was synthesized (Genscript) andcloned into a mammalian expression vector with either a nuclear exportsignal (NES) or nuclear localization sequence (NLS) under expression bythe EF1α promoter. Because of the stability conferred bymonomeric-super-folded GFP (msfGFP), we fused msfGFP to the C-terminusof LwaCas13a. The full-length direct-repeat of LwaCas13a was used forcloning the guide backbone plasmid with expression under a U6 promoter.The catalytically-inactive LwaCas13a-msfGFP construct (dead Cas13a ordCas13a) was generated by introducing R474A and R1046A mutations in thetwo HEPN domains. A drug-selectable version of LwaCas13a-msfGFP wasgenerated by cloning the protein into a backbone with Blasticidinselection marker linked to the C-terminus via a 2A peptide sequence. Thenegative feedback version of the dCas13a-msfGFP construct was generatedby cloning zinc-finger binding site upstream of the promoter ofdCas13a-msfGFP and fusing a Zinc finger and KRAB domain to theC-terminus.

The reporter luciferase construct was generated by cloning Cypridinaluciferase (Cluc) under expression by CMV and Gaussia luciferase (Gluc)under expression by EF1α both on a single vector. Expression of bothluciferases on a single vector allows one luciferase to serve as adosing control for normalization of knockdown of the other luciferase,controlling for variation due to transfection conditions.

For the endogenous knockdown experiments in FIG. 1G, guides and shRNAswere designed using the RNAxs siRNA design algorithm³⁷. The predictiontool was used to design shRNAs and guides were designed in the samelocation to allow for comparison between shRNA and Cas13a knockdown.

The rice actin promoter (pOsActin) was PCR amplified from pANIC6A⁸⁸ andeach Cas13a was PCR amplified from existing Cas13a constructs. Thesefragments were ligated into existing plant expression plasmids such thateach Cas13a was driven by the rice actin promoter and transcription wasterminated by the HSP terminator. Cas13a gRNAs were expressed from therice U6 promoter (pOsU6). The gRNA target sequence was identical foreach gene whereas the scaffold sequence was Cas13a-specific. In theseexperiments, we targeted the rice 5-enolpyruvylshikimate-3-phosphatesynthase (OsEPSPS) gene, which is the target of glyphosate-basedherbicides, and the rice hydroxycinnamoyl-CoA shikimate/quinatehydroxycinnamoyl transferase (OsHCT) gene, which is necessary for properplant growth.

All guides and shRNAs used in this study are listed in SupplementaryTables 2 and 3.

Protoplast Preparation

Green rice protoplasts (Oryza sativa L. ssp. japonica var. Nipponbare)were prepared as previously described⁸⁰ with slight modifications.Seedlings were grown for 14 days and protoplasts resuspended in MMGbuffer containing 0.1M CaCl₂. This modified MMG buffer was used toprepare fresh 40% PEG buffer as well as in place of WI buffer. Finally,protoplasts were kept in total darkness for 48 hourspost-transformation. All other conditions were as previously described.

Nucleic Acid Target and crRNA Preparation for In Vitro Reactions andCollateral Activity

For generation of nucleic acid targets, oligonucleotides were PCRamplified with KAPA Hifi HotStart t (Kapa Biosystems). dsDNA ampliconswere gel extracted and purified using MinElute gel extraction kit(Qiagen). The resulting purified dsDNA was transcribed via overnightincubation at 30° C. with the HiScribe T7 Quick High Yield RNA Synthesiskit (New England Biolabs). Transcribed RNA was purified using theMEGAclear Transcription Clean-up kit (Thermo Fisher). All RNA targetsused in this study are listed in Supplementary Table 4 and 6.

To generate crRNAs, oligonucleotides were ordered as DNA (Integrated DNATechnologies) with an additional 5′ T7 promoter sequence. crRNA templateDNA was annealed with a T7 primer (final concentrations 10 μM) andtranscribed via overnight incubation at 37° C. with the HiScribe T7Quick High Yield RNA Synthesis kit (New England Biolabs). The resultingtranscribed crRNAs were purified with RNAClean XP beads (BeckmanCoulter), using a 2×ratio of beads to reaction volume, supplemented withadditional 1.8×ratio of isopropanol (Sigma). crRNA constructs used forin vitro experiments study are listed in Supplementary Table 5 and crRNAconstructs used for collateral detection are listed in SupplementaryTable 6.

LwaCas13a Cleavage and Collateral Activity Detection

For biochemical characterization of LwaCas13a, assays were performed aspreviously described⁵⁸. Briefly, nuclease assays were performed with 160nM of end-labeled ssRNA target, 200 nM purified LwaCas13a, and 100 nMcrRNA, unless otherwise indicated. All assays were performed in nucleaseassay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). For arrayprocessing, 100ng of in vitro transcribed array was used per nucleaseassay. Reactions were allowed to proceed for 1 hour at 37° C. (unlessotherwise indicated) and were then quenched with proteinase buffer(proteinase K, 60 mM EDTA, and 4M Urea) for 15 minutes at 37° C. Thereactions were then denatured with 4.5M urea denaturing buffer at 95° C.for 5 minutes. Samples were analyzed by denaturing gel electrophoresison 10% PAGE TBE-Urea (Invitrogen) run at 45° C. Gels were imaged usingan Odyssey scanner (LI-COR Biosciences).

Collateral activity detection assays were performed as previouslydescribed⁹⁰. Briefly, reactions consisted of 45 nM purified LwCas13a,22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2,Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs),100 ng of background total human RNA (purified from HEK293FT culture),and varying amounts of input nucleic acid target, unless otherwiseindicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mMMgCl2, pH 7.3). Reactions were allowed to proceed for 1-3 hr at 37° C.(unless otherwise indicated) on a fluorescent plate reader (BioTek) withfluorescent kinetics measured every 5 min.

RNA Extraction and qRT-PCR

Total RNA was isolated after 48 hours of incubation using Trizol and theaccompanying protocol. One nanogram of total RNA was used in theSuperScript III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen)using the accompanying protocol. All samples were run in technicaltriplicate of three biological replicates in a 384-well format on aLightCycler 480 Instrument (Roche). All PCR primers were verified asbeing specific based on melting curve analysis and are as follows:OsEPSPS (Os06 g04280), 5′-TTG CCA TGA CCC TTG CCG TTG TTG-3′ (SEQ ID NO:181) and 5′-TGA TGA TGC AGT AGT CAG GAC CTT-3′ (SEQ ID NO: 182); OsHCT(Os11 g07960), 5′-CAA GTT TGT GTA CCC GAG GAT TTG-3′ (SEQ ID NO: 183)and 5′-AGC TAG TCC CAA TAA ATA TGC GCT-3′ (SEQ ID NO: 184); OsEF1α (Os03g08020), 5′-CTG TAG TCG TTG GCT GTG GT-3′ (SEQ ID NO: 185) and 5′-CAGCGT TCC CCA AGA AGA GT-3′ (SEQ ID NO: 186). Primers for OsEF1α werepreviously described⁹¹. All data are presented as the mean plus/minusthe standard error with each sample relative to the expression of EF1a.

Cloning of Tiling Guide Screens

For tiling guide screens, spacers were designed to target mRNAtranscripts at even intervals to fully cover the entire length of thetranscript. Spacers were ordered from IDT, annealed, and golden-gatecloned into LwaCas13a guide expression constructs with either atRNA^(val) promoter, for Gluc and Cluc screens, or U6 promoter, for allendogenous screens.

Mammalian Cell Culture and Transfection for Knockdown with LwaCas13a

All mammalian cell experiments were performed in the HEK293FT line(ATCC) unless otherwise noted. HEK293FT cells were cultured inDulbecco's Modified Eagle Medium with high glucose, sodium pyruvate, andGlutaMAX (Thermo Fisher Scientific) supplemented with 10% fetal bovineserum (VWR Seradigm) and 1× Penicillin-Streptomycin (Thermo FisherScientific). Cells were passaged to maintain confluency below 70%. Forexperiments involving A375 (ATCC), cells were cultured in RPMI 1640Medium (Thermo Fisher Scientific) supplemented with 9% fetal bovineserum (VWR Seradigm) and 1× Penicillin-Streptomycin (Thermo FisherScientific).

To test knockdown of endogenous genes, Lipofectamine 2000 (Thermo FisherScientific) transfections were performed with 150 ng of LwaCas13aplasmid and 250 ng of guide plasmid per well, unless otherwise noted.Experiments testing knockdown of reporter plasmids were supplementedwith 12.5 ng reporter construct per well. 16 hours prior totransfection, cells were plated in 96-well plates at approximately20,000 cells/well and allowed to grow to 90% confluency overnight. Foreach well, plasmids were combined with Opti-MEM® I Reduced Serum Medium(Thermo Fisher) to a total of 25 μL, and separately 0.5 μL ofLipofectamine 2000 was combined with 24.5 μL of Opti-MEM. Plasmid andlipofectamine solutions were then combined, incubated for 5 min, andslowly pipetted onto cells to prevent disruption.

Transformation of Green Rice Protoplasts

For the green rice experiments, plasmids expressing each Cas13a and thecorresponding gRNA were mixed in equimolar ratios such that a total of30 mg of DNA was used to transform a total of 200,000 protoplasts pertransformation.

Measurement of Luciferase Activity

We harvested media containing secreted luciferase at 48 hours posttransfection, unless otherwise noted. Media was diluted 1:5 in PBS andthen luciferase activity was measured using the NEB Cypridina andGaussia luciferase measurement kits on a Biotek Synergy 4 plate readerwith an injection protocol. All replicates were performed as biologicalreplicates.

Harvest of total RNA and Quantitative PCR

48 hours post-transfection, cells harvesting and reverse transcriptionfor cDNA generation was performed using a previously describedmodification⁹² of the commercial Cells-to-Ct kit (Thermo FisherScientific). Transcript expression was then quantified with qPCR usingFast Advanced Master Mix (Thermo Fisher Scientific) and TaqMan qPCRprobes (Thermo Fisher Scientific, Supplementary Table 7 and 8) withGAPDH control probes (Thermo Fisher Scientific). All qPCR reactions wereperformed in 5 μL reactions with 4 technical replicates in 384-wellformat, and read out using a LightCycler 480 Instrument II (Roche). Formultiplexed targeting reactions, readout of different targets wasperformed in separate wells.

Expression levels were calculated by subtracting housekeeping control(GAPDIH) Ct values from target Ct values to normalize for total input,resulting in ΔCt levels. Relative transcript abundance was computed as2{circumflex over ( )}(−ΔCt). All replicates were performed asbiological replicates.

Computational Analysis of Target Accessibility

To first analyze target accessibility, we analyzed top guides from thetiling screen and determined whether they grouped closer together thanexpected with the assumption that if there were regions ofaccessibility, you would expect multiple guides in that region to behighly active. We defined top guides as the top 20% performing guidesfor the Gluc tiling screen and top 30% performing guides for the Cluc,KRAS, and PPIB tiling screens. We generated a null probabilitydistribution for pair-wise distances between guides by randomlysimulated 10,000 guide positions and then compared the experimentallydetermined top guide pair-wise distances.

Accessibility was predicted using the RNAplfold algorithm in the ViennaRNA software suite⁹³. The default window size of 70 nt was used andprobability of a target region being unpaired was calculated as theaverage of the 28 single-nucleotide unpaired probabilities across thetarget region. These accessibility curves were smoothened and comparedto smoothened knockdown curves across each of the four transcripts andcorrelations between the two factors were computed using Pearson'scorrelation coefficient. The probability space of these two factors wasalso visualized by performing 2D kernel density estimation across thetwo variables.

RNA Sequencing and Analysis

To determine the specificity of LwaCas13a knockdown, we performed RNAsequencing on mRNA from knockdown experiments involving both LwaCas13aand shRNA constructs. Total RNA was prepared from transfectionexperiments after 48 hours using the Qiagen RNeasy Plus Mini kit. mRNAwas then extracted using the NEBNext® Poly(A) mRNA Magnetic IsolationModule and RNA-seq libraries were prepared using the NEBNext® Ultra™Directional RNA Library Prep Kit for Illumina®. RNA-sequencing librarieswere sequenced on an Illumina NextSeq instrument with at least 10M readsper library.

An index was generated using the RefSeq GRCh38 assembly and reads werealigned and quantified using Bowtie and RSEM v1.2.31 using defaultparameters⁹⁴. Transcript per million (TPM) values were used forexpression counts and were transformed to log-space by taking thelog₂(TPM+1).

To find differentially expressed genes, we performed Student's t-test onthe three targeting replicates versus the three non-targetingreplicates. The statistical analysis was only performed on genes thathad a log₂(TPM+1) value greater than 2.5 in at least two of the 6replicates. Only genes that had a differential expression greater than 2or less than 0.75 and a false discovery rate <0.10 were reported to besignificantly differentially expressed.

Cross-correlations between replicates and averages of replicates wereperformed using Kendall's tau coefficient. The variation of shRNA vsCas13a libraries was analyzed by considering the distribution ofstandard deviations for gene expression across the 6 replicates (3targeting and 3 non-targeting replicates) and plotted as violin plots.

Evaluation LwaCas13a for Collateral Activity in Mammalian Cells

LwaCas13a was additionally evaluated for collateral activity by testingfor any potential growth restriction effects. Mammalian cells weretransfected with luciferase reporter target, guide plasmid, and eitherLwaCas13a or drug-selectable LwaCas13a. 24 hours post-transfection,cells were split 1:5 into fresh media and drug-selectable LwaCas13asamples were supplemented with 10 μg/mL Blasticidin S (Thermo FisherScientific). After 48 hours of additional growth, cells were assayed forluciferase knockdown, maintenance of LwaCas13a expression via GFPfluorescence measurement on a multimode plate reader (Biotek Neo2), andcell growth by CellTiter-Glo® Luminescent Cell Viability Assay(Promega).

Quantifying dCas13a Binding with RIP

For RNA immunoprecipitation experiments, HEK293FT cells were plated in6-well plates and transfected with 1.3 μg of dCas13a expression plasmidand 1.7 μg of guide plasmid, with an additional 150 ng of reporterplasmid for conditions involving reporter targeting. 48 hours posttransfection, cells were washed twice with ice-cold PBS (Sigma) andfixed with 0.2% paraformaldehyde (Electron Microscopy Sciences) in PBSfor 15 min at room temperature. After fixation, the paraformaldehyde wasremoved, 125 mM glycine in PBS was added to quench crosslinking, and thecells were incubated for 10 minutes. Cells were washed twice again withice-cold PBS, harvested by scraping, and the cell suspension wascentrifuged at 800 g for 4 min to pellet the cells. The supernatant wasremoved and the pellet was washed with PBS prior to lysis. Cells werelysed with 200 μg of 1×RIPA Buffer (Cell Signaling) supplemented withcOmplete™ ULTRA Tablets, EDTA-free (Sigma) and Ribonuclease inhibitor(Sigma R1158). Cells were allowed to lyse on ice for 10 min and thensonicated for 2 min with a 30 sec on/30 sec off cycle at low intensityon a Bioruptor sonicator (Diagenode). Insoluble material pelleted bycentrifugation at 16,000 g for 10 min at 4° C., and the supernatantcontaining cleared lysate was used for pulldown with magnetic beads.

To conjugate antibodies to magnetic beads, 100 μL/sample of Dynabeads®Protein A for Immunoprecipitation (Thermo Fisher Scientific) werepelleted by application of a magnet, and the supernatant was removed.Beads were resuspended in 200 μL of wash buffer (PBS supplemented with0.02% Tween-20 (Sigma)) and 5 μg of rabbit Anti-Mouse IgG (Sigma M703)was added. The sample was incubated for 10 min at room temperature on arotator to allow antibody to conjugate to the beads. After incubation,beads were pelleted via magnet, supernatant was removed, and beads werewashed twice with wash buffer. The pellet was resuspended in 100 μL washbuffer and split into two 50 μL volumes for conjugation of Anti-HAantibody (Thermo Fisher Scientific 26183) or IgG antibody control (SigmaI5381). For each antibody, 2.5 μg of antibody was added with 200 μL washbuffer and incubated for 10 min at room temperature on a rotator.Post-incubation, beads were pelleted via magnet and washed twice withwash buffer, and resuspended in 200 μL 1×RIPA with Ribonucleaseinhibitor (Sigma R1158) and protease inhibitor cocktail (Sigma P8340).100 μL of sample lysate was added to beads and rotated overnight at 4°C.

After incubation with sample lysate, beads were pelleted, washed threetimes with 1×RIPA, 0.02% Tween-20, and then washed with DNase buffer(350 mM Tris-HCl [pH 6.5]; 50 mM MgCl2; 5 mM DTT). Beads wereresuspended in DNase buffer and TURBO DNase (Life Technologies) wasadded to final concentration of 0.08 units/μl. DNase was incubated 30min at 370 C on a rotator. Proteins were then digested by addition ofProteinase K (New England Biosciences) to a final concentration of 0.1units/μl and incubated at 37° C. with rotation for an additional 30 min.For denaturation and purification, urea (Sigma) was added to a finalconcentration of 2.5 M, samples were incubated for 30 min, and RNA waspurified using a Direct-Zol RNA miniprep (Zymo Research). Purified RNAwas reverse transcribed to cDNA using the qScript Flex cDNA (Quantabio)and pulldown was quantified with qPCR using Fast Advanced Master Mix andTaqMan qPCR probes (Supplementary Table 7 and 8). All qPCR reactionswere performed in 5 μL reactions with 4 technical replicates in 384-wellformat, and read out using a LightCycler 480 Instrument II. Enrichmentwas quantified for samples as compared to their matched IgG antibodycontrols.

Translocation Measurement of LwaCas13a and LwaCas13a-NF

HEK293FT cells were plated in 24-well tissue culture plates onpoly-D-lysine coverslips (Corning) and transfected with 150ng dCas13a-NFvector and 300ng guides for imaging ACTB. For translocation experiments,cells were fixed with 4% PFA and permeabilized with 0.2% Triton X-100after 48 hours and mounted using antifade mounting medium with DAPI(Vectashield). Confocal microscopy was performed using a Nikon EclipseTi1 with Andor Yokogawa Spinning disk Revolution WD system.

Nuclear export of dCas13a-NF-msfGFP with guides targeting ACTB mRNA wasanalyzed by measuring the average cytoplasmic and nuclear msfGFPfluorescence and comparing the ratio across many cells between targetingand non-targeting conditions.

Fluorescent In Situ Hybridization (FISH) of ACTB Transcript

HEK293FT cells were plated in 24-well tissue culture plates onpoly-D-lysine coverslips (Corning) and transfected with 75ng dCas13a-NFvector and 250ng guides for imaging ACTB. After 48 hours, cells werefixed with 4% PFA for 45 minutes. The QuantiGene viewRNA ISH Cell assaykit (Affymetrix) was used for performing the FISH on the cell samplesand the protocol was followed as described by the manufacturer. Afterfinishing the FISH procedure, coverslips were mounted using antifademounting medium (Vectashield). Confocal microscopy was performed using aNikon Eclipse Ti1 with Andor Yokogawa Spinning disk Revolution WDsystem.

Tracking of LwaCas13a to Stress Granules

HEK293FT cells were plated in 24-well tissue culture plates onpoly-D-lysine coverslips (Corning) and transfected with 75ng dCas13a-NFvector and 250ng guides for imaging ACTB. For stress granuleexperiments, 200 μM sodium arsenite was applied for 1 hour prior tofixing and permeabilizing the cells. For immunofluorescence of G3BP1,cells were blocked with 20% goat serum, and incubated over night at roomtemperature with anti-G3BP1 primary antibody (Abnova H00010146-B01P).Cells were then incubated with secondary antibody labeled with AlexaFluor 594 and mounted using anti-fade mounting medium with DAPI(Vectashield). Confocal microscopy was performed using a Nikon EclipseTi1 with Andor Yokogawa Spinning disk Revolution WD system.

Stress granule co-localization with dCas13a-NF-msfGFP was calculatedusing the average msfGFP and G3BP1 signal per cell using Pearson'scorrelation coefficient. The colocalization analyses were performed inthe image analysis software FIJI⁹⁵ using the Coloc 2 plugin.

For live imaging experiments, HEK293FT cells were plated in 96-welltissue culture plates and transfected with 150ng dCas13a-NF vector,300ng guides for imaging ACTB, and 5ng of G3BP1-RFP reporter. After 48hours, the cells were subjected to 0 μM or 400 μM sodium arsenite andimaged every 15 minutes every 2 hours on an Opera Phenix™ High ContentScreening System (PerkinElmer) using the spinning disk confocal settingwith 20×water objective. Cells were maintained at 37° C. in a humidifiedchamber with 50% CO₂. Live cell dCas13a-NF-msfGFP colocalization withG3BP1-RFP in stress granules was measured using the Opera Phenix Harmonysoftware (PerkinElmer).

Correlation of Target Accessibility and Knockdown

We showed that LshCas13a targeted the MS2 ssRNA genome in definedlocations with highly effective guides grouping together closer thanwould be expected by chance¹. We hypothesized that clusters of effectiveguides could arise from regions of accessibility being better targetedby LshCas13a due to sterics and availability of target binding. Wesought to analyze whether these results would extend to targeting ofmammalian mRNAs with LwCas13a and so performed a similar analysis on thefour genes we tiled with guides: Gluc, Cluc, KRAS, and PPIB. We firstdefined effective guides as the top 20% of guides for Gluc and top 30%of guides for the other three genes as measured by knockdown efficiency(note that we are more generous with the threshold on Cluc, KRAS, andPPIB because they were tiled with 50% fewer guides than Gluc). Inanalyzing only the most effective guides as defined using thesethresholds, we found that they do cluster closer together than expectedby chance.

We next sought to model the accessibility of the target transcript usingthe RNAplfold algorithm from the Vienna RNA software suite. Wecalculated the accessibility across windows of size 70 (default), andthe probability that a given nucleotide position on the transcript wouldbe unpaired. For a given guide, we can then average the probabilitiesacross the 28 nt to obtain the average probability that a given guidetarget is unpaired and thus accessible. We take this calculation as ameasure for target accessibility and compare against the targetexpression for each guide. Because of the variation that occur fromguide to guide, especially because the genes are not densely tiled, wedecided to smooth both the target accessibility and target expressioncurves. By plotting these curves and analyzing the correlation, we foundthat target accessibility is significantly correlated to targetexpression and that it can explain 4.4%-16% of the variation in targetexpression. To offset spurious effects of the smoothing, we alsoanalyzed the probability versus knockdown data in probability space byusing kernel density estimation. This analysis revealed a similarpositive relationship between the probability of a target region beingbase-paired and target expression. Finally, to offset anyparameter-specific aspects of RNAplfold yielding spurious correlationsfor the base-pairing probability, we calculated the unpaired probabilityfor a grid of parameters: window size and k-mer size. We initially onlycalculated the probability for k-mers of 1, i.e. probability that agiven position is unpaired averaged across the 28 nt target region. Withk=2, we calculate the probability pairs of nucleotides are unpaired andaverage those across the 28 nt region and so on for k up to 28. Thisanalysis revealed regions of parameters for each transcript yieldingpositive correlations which provides more confidence that it's not justa specific parameter set giving a correlation. As might be expected, alarger transcript such as KRAS has positive correlations for largerwindow sizes versus smaller transcripts such as PPIB, which had positivecorrelations for much smaller window sizes.

Comparison of Target Knockdown Specificity Between shRNA and Cas13a

For rigorous comparison between shRNA and Cas13a transcript knockdownspecificity, we wanted to evaluate the transcriptome-wide effects ofperturbation compared between targeting and non-targeting controls.Off-targets will appear as deviations from the identity line, and it isapparent that more off-targets occur for shRNA than for Cas13a whentargeting reporter constructs. Similar increased deviation occurs forendogenous genes. While it may be expected that knockdown of endogenoustranscripts would result in additional gene expression changes fromchanges in biological function, we find that PPIB and KRAS knockdownwith Cas13a does not result in expression changes, possibly because thelevel of knockdown is not adequate for substantial downstream effects.

Next, we quantified the number of off-targets via differential geneexpression: off-target transcripts were defined as genes with at least100% up-regulation or 25% down-regulation. Significant off-targets werethen compared between targeting and non-targeting conditions byStudent's t-test with multiple comparisons corrected viaBenjamini-Hochberg procedure with an FDR of 0.1. We found that for allthree transcripts tested, there were hundreds of significant off-targettranscripts as a result of shRNA targeting, but no significantoff-targets as a result of Cas13a targeting.

The large variation in shRNA perturbations between targeting andnon-targeting conditions could either be due to increased overall geneexpression variation, which would result in similar deviations betweenbiological replicates, or reproducible and consistent off targets, whichwould manifest as increased correlations between replicates for the samecondition. Comparing the non-targeting and targeting replicates to eachother for Gluc perturbation, we found little variation betweenreplicates. However, when we compared individual replicates of targetingconditions versus all non-targeting conditions for either shRNA orCas13a, we found that comparisons between replicates recapitulated theoff-targets seen for mean measurements. To quantify the correlationsbetween replicates, we compared all replicates for targeting andnon-targeting conditions across reporter and endogenous genes. shRNAsamples correlated higher within replicates than between non-targetingand targeting conditions, while correlations between Cas13a samples hadless variation between targeting and non-targeting perturbations. Wethen compared all samples between each other and found that shRNAsamples had lower correlation between samples and with Cas13a thanwithin Cas13a, showing that Cas13a was more consistent. Lastly, wecalculated the standard deviation per gene across both targeting andnon-targeting replicates, and found that the distribution of standarddeviations was lower for Cas13a perturbations than for shRNAperturbations across the transcriptome. Overall, this evidence showsthat Cas13a targeting results in less transcriptome-wide variation.

Evidence for Lack of Collateral Activity in Mammalian Cells

LshCas13a and LwCas13a seems to display robust collateral activity invitro. Because of concerns about cell health and the specificity ofLwCas13a knockdown due to the collateral effect, we have attempted tostudy whether the collateral effect is in fact present in mammaliancells. Throughout our manuscript, we collect data that suggests a lackof collateral activity for LwCas13a, and here, we gather all the piecesof evidence and discuss them in detail.

1. RNA sequencing: We performed RNA sequencing to determine thespecificity of LwCas13a knockdown and found that LwCas13a displayed muchgreater specificity than RNAi. In our differential expression analysis,there were no significantly differentially expressed off-targets for anyLwCas13a condition analyzed, despite significant knockdown of the targettranscript. Assuming that there was not uniform knockdown of everytranscript in the human transcriptome, we believe that this offerssubstantial evidence that knockdown by LwCas13a is specific and there isno collateral effect in mammalian cells.

2. Tiling screens: In the four gene tiling screens we performed, wefailed to see knockdown of the control genes used for normalization.This is a similar analysis to the RNA sequencing, although focused onjust one other gene rather than the transcriptome. This result isimportant, however, because we fail to see collateral activity acrosshundreds of guides tested, providing confidence that LwCas13a robustlyshows no collateral activity in vivo.

3. Leave one out multiplexing: To ascertain the specificity of geneknockdown in our multiplexing experiments, we designed an experimentwhere each gene-targeting guide is replaced by a non-targeting guide ina guide expression array containing guides against three differentgenes. We found that there was no significant change in gene expressionfor the gene targeted by the missing guide. We believe this is also anice demonstration that there is a lack of collateral activity inmammalian cells because we would expect all three genes to always showknockdown despite whether its guide is present or missing.

4. Growth experiment: Previously, we showed that LshCas13a caused growthrestriction in bacteria during gene knockdown¹. We designed astraightforward experiment to measure whether there was growth effectsof knockdown in mammalian cells. We allowed cells with LwCas13atargeting Gluc to grow for 72 hours and then measured cell viability andfound that there was no difference in growth between targeting andnon-targeting guide conditions. We additionally controlled for LwCas13aexpression by looking at fluorescence of a C-terminal msfGFP fusion andshowed that expression was the same across all conditions and notselected out due to a potentially deleterious phenotype. There are manypossible reasons for a lack of growth inhibition in mammalian cellsdespite previous observations of bacterial growth suppression, includingdifferences in the RNA cytoplasmic density, cytosol composition, andmechanisms for processing of cleaved transcripts.

Points 1-3 collectively show that targeted gene knockdown does notaffect other genes beyond the targeted gene and Point 4 reveals thatthere is no observable side effects for gene knockdown. We believe thatthis data all together is substantial evidence for a lack of LwCas13acollateral activity in mammalian cells and that Cas13 knockdown is quitespecific.

Importance of Negative-Feedback Construct for dCas13a Imaging

For live-cell imaging, it is important to maintain a high signal tonoise ratio and reduce background. The concern of background isespecially important for fluorophore-labeled constructs, such asdCas13a, as unbound dCas13a cannot be removed from the cell ordistinguished from bound protein. One approach to reducing background issequestration of the protein in the nucleus via nuclear localizationsignal (NLS) tags; upon nascent transcript binding, the protein istransiently exported to the cytoplasm. Although this technique has beenutilized in MS2 systems, we found that it was imperfect due to theoff-target nuclear leakage and on-target nuclear escape. To improve uponthe signal to noise of NLS-tagging alone, we incorporated a negativefeedback system where nuclear resident protein will inhibit furtherexpression of dCas13a. We find that negative feedback regulation reducesspurious translocation to the cytoplasm and leads to reduced overalllevels of dCas13a-NF levels in non-targeting conditions compared totargeting conditions, thereby increasing the utility of dCas13a as animaging tool.

Supplementary Tables

SUPPLEMENTARY TABLE 1 Plasmids used in this study. Plasmid NameDescription pC004 beta-lactamase screening target pC014 LwaCas13a-msfGFPpC015 dLwaCas13a-NF pC016 LwaCas13a guide expression backbone with U6promoter pC017 LwaCas13a guide expression backbone with tRNAval promoterpC018 LshCas13a from Leptotrichia shahii pC019 LwaCas13a fromLeptotrichia wadei pC020 LseCas13a from Listeria seeligeri pC021LbmCas13a from Lachnospiraceae bacterium MA2020 pC022 LbnCas13a fromLachnospiraceae bacterium NK4A179 pC023 CamCas13a from [Clostridium]aminophilum DSM 10710 pC024 CgaCas13a from Carnobacterium gallinarum DSM4847 pC025 Cga2Cas13a from Carnobacterium gallinarum DSM 4847 pC026PprCas13a from Paludibacter propionicigenes WB4 pC027 LweCas13a fromListeria weihenstephaensis ESL R9-0317 pC028 LbfCas13a from Listeriaceaebacterium FSL M6-0635 pC029 Lwa2Cas13a from Leptotrichia wadei F0279pC030 RcsCas13a from Rhodobacter capsulatus SB 1003 pC031 RcrCas13a fromRhodobacter capsulatus R121 pC032 RcdCas13a from Rhodobacter capsulatusDE442 pC033 Dual luciferase reporter (Gluc and CluC) pC034LwaCas13a-msfGFP-2A-Blast pC035 dLwaCas13a-msfGFP

SUPPLEMENTARY TABLE 2 Guides used for in vivo experiments in this study.Name SEQ ID NO: Guide sequence PFS PFS targeting spacer 187AGATTGCTGTtctaccaagtaatc N/A cata PFS non-targeting spacer 188tatggattacttggtagaACAGCAA N/A TCT Glue guide 1 189 ATCAGGGCAAACAGAAC CTTTGACTCCca Glue guide 2 190 GTGCAGCCAGCTTTCCGG C GCATTGGCTTNon-targeting guide 191 tagattgctgttctaccaagtaatccat N/APPIB RNAxs guide 1 192 tccttgattacacgatggaatttgctgt CCXCR4 RNAxs guide 1 193 atgataatgcaatagcaggacaggatga CKRAS RNAxs guide 1 194 aatttctcgaactaatgtatagaaggca C EPSPS guide 1 195CCACCACCACCGCCTCCCGCCG C CCCCCG EPSPS guide 2 196TGCTCCCATCATCTCAAGTACCT A CAGCA EPSPS guide 3 197 CCCTTGACACGAACAGGTGGGA CATTCAG HCT guide 1 198 AGAAGGTCACCTGTACGGCGA T GCACGGC HCT guide 2199 CAGATCCGCTTGAGGGTGGCG C ATCTGGT HCT guide 3 200CCGGACGATCGGGCATCCCCG A CCATCTC PDS guide 1 201 GACTGAGCACAAAGCTTCCCA TGATAGAA PDS guide 2 202 ACCATCCAAGAATGCCATCTTA A GAACCA PDS guide 3 203CCTGGCAAACAACCTGTAGAG A CACCGAG Non-targeting guide for 204TAGATTGCTGTTTCACACAGAT N/A green rice protoplast ATGCAT experimentKRAS top guide 1 205 tataatggtgaatatcttcaaatgattt G KRAS top guide 2 206atgtatagaaggcatcatcaacaccctg U KRAS top guide 3 207ggttaaaaatttacagattgtgctgagc U PPIB top guide 1 208gtagatgctctttcctcctgtgccatct G PPIB top guide 2 209cagtttgaagttctcatcggggaagcgc A PPIB top guide 3 210cagtgttggtaggagtttgttacaaaag A MALAT1 top guide 1 211 CTTGGCCAAGTCTGTTATC GTTCACCTGA MALAT1 top guide 2 212 CAAAATGTACTCAGCTTC C AATCACAAATMALAT1 top guide 3 213 GGTTATAGCTTGACAAGC A AATTAACTTTPPIB multiplexing guide 214 tccttgattacacgatggaatttgctgt CCXCR4 multiplexing guide 215 atgataatgcaatagcaggacaggatga CKRAS multiplexing guide 216 aatttctcgaactaatgtatagaaggca CTINCR multiplexing guide 217 gcgtgagccaccgcgcctggccggctgt CPCAT multiplexing guide 218 ccagctgcagatgctgcagtttttggcg C gLuc1_WT 219ATCAGGGCAAACAGAAC C TTTGACTCCCA gLuc1_1 220 TTCAGGGCAAACAGAAC CTTTGACTCCCA gLuc1_4 221 ATCTGGGCAAACAGAAC C TTTGACTCCCA gLuc1_7 222ATCAGGCCAAACAGAAC C TTTGACTCCCA gLuc1_10 223 ATCAGGGCATACAGAAC CTTTGACTCCCA gLuc1_13 224 ATCAGGGCAAACTGAAC C TTTGACTCCCA gLuc1_16 225ATCAGGGCAAACAGATC C TTTGACTCCCA gLuc1_19 226 ATCAGGGCAAACAGAAC CTATGACTCCCA gLuc1_22 227 ATCAGGGCAAACAGAAC C TTTGTCTCCCA gLuc1_25 228ATCAGGGCAAACAGAAC C TTTGACTGCCA gLuc1_28 229 ATCAGGGCAAACAGAAC CTTTGACTCCCT CXCR4_WT 230 ATGATAATGCAATAGCA C GGACAGGATGA CXCR4_1 231TTGATAATGCAATAGCAG C GACAGGATGA CXCR4_4 232 ATGTTAATGCAATAGCAG CGACAGGATGA CXCR4_7 233 ATGATATTGCAATAGCAG C GACAGGATGA CXCR4_10 234ATGATAATGGAATAGCA C GGACAGGATGA CXCR4_13 235 ATGATAATGCAAAAGCA CGGACAGGATGA CXCR4_16 236 ATGATAATGCAATAGGA C GGACAGGATGA CXCR4_19 237ATGATAATGCAATAGGA C GCACAGGATGA CXCR4_22 238 ATGATAATGCAATAGCA CGGACTGGATGA CXCR4_25 239 ATGATAATGCAATAGCA C GGACAGGTTGA CXCR4_28 240ATGATAATGCAATAGCA C GGACAGGATGT gLuc3_WT 241 GTGCAGCCAGCTTTCCGG CGCATTGGCTT gLuc3_1 242 CTGCAGCCAGCTTTCCGG C GCATTGGCTT gLuc3_4 243GTGGAGCCAGCTTTCCGG C GCATTGGCTT gLuc3_7 244 GTGCAGGCAGCTTTCCGG CGCATTGGCTT gLuc3_10 245 GTGCAGCCACCTTTCCGG C GCATTGGCTT gLuc3_13 246GTGCAGCCAGCTATCCGG C GCATTGGCTT gLuc3_16 247 GTGCAGCCAGCTTTCGGG CGCATTGGCTT gLuc3_19 248 GTGCAGCCAGCTTTCCGG C CCATTGGCTT gLuc3_22 249GTGCAGCCAGCTTTCCGG C GCAATGGCTT gLuc3_25 250 GTGCAGCCAGCTTTCCGG CGCATTGCCTT gLuc3_28 251 GTGCAGCCAGCTTTCCGG C GCATTGGCTAKRAS_top_tiling_WT 252 TATAATGGTGAATATCTT G CAAATGATTT KRAS_top_tiling_1253 AATAATGGTGAATATCTT G CAAATGATTT KRAS_top_tiling_4 254TATTATGGTGAATATCTT G CAAATGATTT KRAS_top_tiling_7 255 TATAATCGTGAATATCTTG CAAATGATTT KRAS_top_tiling_10 256 TATAATGGTCAATATCTT G CAAATGATTTKRAS_top_tiling_13 257 TATAATGGTGAAAATCTT G CAAATGATTTKRAS_top_tiling_16 258 TATAATGGTGAATATGTT G CAAATGATTTKRAS_top_tiling_19 259 TATAATGGTGAATATCTT G GAAATGATTTKRAS_top_tiling_22 260 TATAATGGTGAATATCTT G CAATTGATTTKRAS_top_tiling_25 261 TATAATGGTGAATATGTT G CAAATGTTTTKRAS_top_tiling_28 262 TATAATGGTGAATATCTT G CAAATGATTAPPIB_top_tiling_WT 263 GTAGATGCTCTTTCCTCC G TGTGCCATCT PPIB_top_tiling_1264 CTAGATGCTCTTTCCTCC G TGTGCCATCT PPIB_top_tiling_4 265GTACATGCTCTTTCCTCC G TGTGCCATCT PPIB_top_tiling_7 266 GTAGATCCTCTTTCCTCCG TGTGCCATCT PPIB_top_tiling_10 267 GTAGATGCTGTTTCCTCC G TGTGCCATCTPPIB_top_tiling_13 268 GTAGATGCTCTTACCTCC G TGTGCCATCTPPIB_top_tiling_16 269 GTAGATGCTCTTTCCACC G TGTGCCATCTPPIB_top_tiling_19 270 GTAGATGCTCTTTCCTCC G AGTGCCATCTPPIB_top_tiling_22 271 GTAGATGCTCTTTCCTCC G TGTCCCATCTPPIB_top_tiling_25 272 GTAGATGCTCTTTCCTCC G TGTGCCTTCTPPIB_top_tiling_28 273 GTAGATGCTCTTTCCTCC G TGTGCCATCA gLuc3_WT 274GTGCAGCCAGCTTTCCGG C GCATTGGCTT gLuc3_double_consec_1 275CAGCAGCCAGCTTTCCGG C GCATTGGCTT gLuc3_double_consec_4 276GTGGTGCCAGCTTTCCGG C GCATTGGCTT gLuc3_double_consec_7 277GTGCAGGGAGCTTTCCGG C GCATTGGCTT gLuc3_double_consec_10 278GTGCAGCCACGTTTCCGG C GCATTGGCTT gLuc3_double_consec_13 279GTGCAGCCAGCTAACCG C GGCATTGGCTT gLuc3_double_consec_16 280GTGCAGCCAGCTTTCGCG C GCATTGGCTT gLuc3_double_consec_19 281GTGCAGCCAGCTTTCCGG C CGATTGGCTT gLuc3_double_consec_22 282GTGCAGCCAGCTTTCCGG C GCAAAGGCTT gLuc3_double_consec_25 283GTGCAGCCAGCTTTCCGG C GCATTGCGTT gLuc3_double_nonconsec_1 284CTGCTGCCAGCTTTCCGG C GCATTGGCTT gLuc3_double_nonconsec_2 285CTGCAGCCTGCTTTCCGG C GCATTGGCTT gLuc3_double_nonconsec_3 286CTGCAGCCAGCTATCCGG C GCATTGGCTT gLuc3_double_nonconsec_4 287CTGCAGCCAGCTTTCCCG C GCATTGGCTT gLuc3_double_nonconsec_5 288GTGGAGCGAGCTTTCCGG C GCATTGGCTT gLuc3_double_nonconsec_6 289GTGGAGCCAGCATTCCG C GGCATTGGCTT gLuc3_double_nonconsec_7 290GTGGAGCCAGCTTTCGGG C GCATTGGCTT gLuc3_double_nonconsec_8 291GTGGAGCCAGCTTTCCGG C GGATTGGCTT gLuc3_double_nonconsec_9 292GTGCAGGCAGGTTTCCGG C GCATTGGCTT gLuc3_double_nonconsec_10 293GTGCAGGCAGCTTTGCGG C GCATTGGCTT gLuc3_double_nonconsec_11 294GTGCAGGCAGCTTTCCGG C CCATTGGCTT gLuc3_double_nonconsec_12 295GTGCAGGCAGCTTTCCGG C GCATAGGCTT gLuc3_double_nonconsec_13 296GTGCAGCCACCTTACCGG C GCATTGGCTT gLuc3_double_nonconsec_14 297GTGCAGCCACCTTTCCGC C GCATTGGCTT gLuc3_double_nonconsec_15 298GTGCAGCCACCTTTCCGG C GCAATGGCTT gLuc3_double_nonconsec_16 299GTGCAGCCACCTTTCCGG C GCATTGGGTT gLuc3_double_nonconsec_17 300GTGCAGCCAGCTATCCCG C GCATTGGCTT gLuc3_double_nonconsec_18 301GTGCAGCCAGCTATCCGG C GCTTTGGCTT gLuc3_double_nonconsec_19 302GTGCAGCCAGCTATCCGG C GCATTGCCTT gLuc3_double_nonconsec_20 303GTGCAGCCAGCTTTCGGG C GGATTGGCTT gLuc3_double_nonconsec_21 304GTGCAGCCAGCTTTCGGG C GCATTCGCTT gLuc3_double_nonconsec_22 305GTGCAGCCAGCTTTCGGG C GCATTGGCTA gLuc3_double_nonconsec_23 306GTGCAGCCAGCTTTCCGG C CCATAGGCTT gLuc3_double_nonconsec_24 307GTGCAGCCAGCTTTCCGG C CCATTGGCAT Glue RNA seq guide 308 ATCAGGGCAAACAGAACC TTTGACTCCca KRAS RNA seq guide 309 gctgtaataattaggtaacatttatttc CPPIB RNA seq guide 310 gtcagtgttggtaggagtttgttacaaa CGlue RNA seq guide 2 311 GTGCAGCCAGCTTTCCGG C GCATTGGCTT ACTB guide 1312 ctggcggcgggtgtggacgggcggcg C ga ACTB guide 2 313gagccacacgcagctcattgtagaaggt C

SUPPLEMENTARY TABLE 3 shRNA used in this study. SEQ  ID  Name NO:Guide sequence Glue shRNA 1 314 AAAGUUCUGUUUGCCCUGAUCcucgagGAUCAGGGCAAACAGAACUUU Glue shRNA 2 315 AAGCCAAUGCCCGGAAAGCUGcucgagCAGCUUUCCGGGCAUUGGCUU Non-targeting shRNA 316UAGAUUGCUGUUCUACCAAGUcucgagACU UGGUAGAACAGCAAUCUA PPIB RNAxs shRNA 1 317ACAGCAAAUUCCAUCGUGUAAcucgagUUA CACGAUGGAAUUUGCUGU CXCR4 RNAxs shRNA 1318 AUCCUGUCCUGCUAUUGCAUUcucgagAAU GCAAUAGCAGGACAGGAU KRAS RNAxs shRNA 1319 CCUUCUAUACAUUAGUUCGAGcucgagCUC GAACUAAUGUAUAGAAGG Glue top shRNA 1320 AAGCCAAUGCCCGGAAAGCUGcucgagCAG CUUUCCGGGCAUUGGCUU Glue top shRNA 2321 AGAUUCCUGGGUUCAAGGACUcucgagAGU CCUUGAACCCAGGAAUCU Glue top shRNA 3322 AAAGUUCUGUUUGCCCUGAUCcucgagGAU CAGGGCAAACAGAACUUU Clue top shRNA 1323 AAGUGGCUGGAGACAUCAUUGcucgagCAA UGAUGUCUCCAGCCACUU Clue top shRNA 2324 AAGCCGUGUCCGUCCCGUACAcucgagUGU ACGGGACGGACACGGCUU KRAS top shRNA 1325 AAGACCUUAAUUCUUGCCGUUcucgagAAC GGCAAGAAUUAAGGUCUU KRAS top shRNA 2326 AAUCAUUUGAAGAUAUUCACCcucgagGGU GAAUAUCUUCAAAUGAUU KRAS top shRNA 3327 AGGGUGUUGAUGAUGCCUUCUcucgagAGA AGGCAUCAUCAACACCCU PPIB top shRNA 1328 AAUCUGUAAAUUUUUAACCUAcucgagUAG GUUAAAAAUUUACAGAUU PPIB top shRNA 2329 AGAUGGCACAGGAGGAAAGAGcucgagCUC UUUCCUCCUGUGCCAUCU PPIB top shRNA 3330 AGCGCUUCCCCGAUGAGAACUcucgagAGU UCUCAUCGGGGAAGCGCU MALAT1 top shRNA 1331 AACAUAACAGACUUGGCCAAGcucgagCU UGGCCAAGUCUGUUAUGUU MALAT1 top shRNA 2332 AAGCUGAGUACAUUUUGCUGGcucgagCC AGCAAAAUGUACUCAGCUU MALAT1 top shRNA 3333 AAGUUAAUUGCUUGUCAAGCUcucgagAG CUUGACAAGCAAUUAACUU Gluc RNA seq 334AAAGUUCUGUUUGCCCUGAUCcucgagGAU shRNA 1 CAGGGCAAACAGAACUUUKRAS RNA seq shRNA 335 UGUUACCUAAUUAUUACAGCCcucgagGG CUGUAAUAAUUAGGUAACAPPIB RNA seq shRNA 336 AACUCCUACCAACACUGACCAcucgagUG GUCAGUGUUGGUAGGAGUUGlue RNA seq 337 AACUUCGCGACCACGGAUCUCcucgagGA shRNA 2GAUCCGUGGUCGCGAAGUU

SUPPLEMENTARY TABLE 4 ssRNA targets used in this study SEQ  ID  Name NO:Guide sequence ssRNA 1 338 GGGGGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGGU AGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUAGCUG UUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAG ssRNA 2 339 GGGUAGGUGUUCCACAGGGUAGCCAGCAGCAUCCUGCGAUGCAAAUAUGGAUUACUUGGU AGAACAGCAAUCUAAUCCGGAACAUAAUGGUGCAGGGCGCUGACUUCCGCGUUUCCAGAC UUUACGAAACACGGAAACCGAAGACCAUUCAUGUUGUUGCUGCCGGAAGCAUAAAG ssRNA 3 340 GGGCCCCUCCGUUCGCGUUUACGCGGACGGUGAGACUGAAGAUAAUAUGGAUUACUUGG UAGAACAGCAAUCUAAACUCAUUCUCUUUAAAAUAUCGUUCGAACUGGACUCCCGGUCGU UUUAACUCGACUGGGGCCAAAACGAAACAGUGGCACUACCCCGCCGGAAGCAUAAAG Modified ssRNA 2 U 341GGGUAGGUGUUCCACAGGGUAGCCAGCAGC AUCCUGCGAUGCAAAUAUGGAUUACUUGGUAGAACAGCAAUCUAAUCCGGAACAUAAUGG UGCAGGGCGCUGACUUCCGCGUUUGUUUUAAAUCAAACACGGAAACCGAAGACCAUUCAU GUUGUUGCUGCCGGAAGCAUAAAGModified ssRNA 2 C 342 GGGUAGGUGUUCCACAGGGUAGCCAGCAGCAUCCUGCGAUGCAAAUAUGGAUUACUUGGU AGAACAGCAAUCUAAUCCGGAACAUAAUGGUGCAGGGCGCUGACUUCCGCGUUUGCCCCA AACCAAACACGGAAACCGAAGACCAUUCAUGUUGUUGCUGCCGGAAGCAUAAAG Modified ssRNA 2 G 343GGGUAGGUGUUCCACAGGGUAGCCAGCAGC AUCCUGCGAUGCAAAUAUGGAUUACUUGGUAGAACAGCAAUCUAAUCCGGAACAUAAUGG UGCAGGGCGCUGACUUCCGCGUUUGGGGGAAAGCAAACACGGAAACCGAAGACCAUUCAU GUUGUUGCUGCCGGAAGCAUAAAGModified ssRNA 2 A 344 GGGUAGGUGUUCCACAGGGUAGCCAGCAGCAUCCUGCGAUGCAAAUAUGGAUUACUUGGU AGAACAGCAAUCUAAUCCGGAACAUAAUGGUGCAGGGCGCUGACUUCCGCGUUUGAAAAA AAACAAACACGGAAACCGAAGACCAUUCAUGUUGUUGCUGCCGGAAGCAUAAAG LwCas13a array 345GGGGAUUUAGACUACCCCAAAAACGAAGGG GACUAAAACUUUCUUUUCUUCGAUGUGGAUUGGUUUACCAGGAUUUAGACUACCCCAAAA ACGAAGGGGACUAAAACGUAAAAUAGAUACAAUAACUUCUUCUACAUGAUUUAGACUACC CCAAAAACGAAGGGGACUAAAACgcgcg

SUPPLEMENTARY TABLE 5 Guides used for in vitroexperiments in this study. SEQ  ID  Name NO: Guide sequence crRNA 1 346GGGGATTTAGACTAGCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGTAATCCATcrRNA 1_29 347 GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGATTGCTGTTCTACCAAGTAATCCATA crRNA 1_27 348GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGTAATCCAcrRNA 1_26 349 GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGATTGCTGTTCTACCAAGTAATCC crRNA 1_25 350GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGTAATCcrRNA 1_24 351 GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGATTGCTGTTCTACCAAGTAAT crRNA 1_23 352GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGTAAcrRNA 1_22 353 GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGATTGCTGTTCTACCAAGTA crRNA 1_21 354GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGT crRNA 1_20355 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCAAGcrRNA 1_19 356 GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAACTAGATTGCTGTTCTACCAA crRNA 1_18 357GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACCA crRNA 1_17358 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTACC crRNA 1_16359 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTAC crRNA 1_15360 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCTA crRNA 1_14361 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTCT crRNA 1_13362 GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTTC crRNA 1_12 363GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGTT crRNA 1_11 364GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTGT crRNA 1_10 365GGGGATTTAGACTACCCCAAAAACGAAGGGGAC TAAAACTAGATTGCTG

SUPPLEMENTARY TABLE 6 ssRNA targets and crRNAs used for the SHERLOCKexperiments. SEQ  ID  Name NO: Guide sequenceCas13a Collateral detection 366 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 28 AAAACuagauugcuguucuaccaaguaauccauCas13a Collateral detection 367 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 27 AAAACuagauugcuguucuaccaaguaauccaCas13a Collateral detection 368 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 26 AAAACuagauugcuguucuaccaaguaauccCas13a Collateral detection 369 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 25 AAAACuagauugcuguucuaccaaguaaucCas13a Collateral detection 370 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 24 AAAACuagauugcuguucuaccaaguaauCas13a Collateral detection 371 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 23 AAAACuagauugcuguucuaccaaguaa Cas13a Collateral detection372 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 22AAAACuagauugcuguucuaccaagua Cas13a Collateral detection 373GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 21AAAACuagauugcuguucuaccaagu Cas13a Collateral detection 374GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 20AAAACuagauugcuguucuaccaag Cas13a Collateral detection 375GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 19AAAACuagauugcuguucuaccaa Cas13a Collateral detection 376GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 18AAAACuagauugcuguucuacca Cas13a Collateral detection 377GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 17AAAACuagauugcuguucuacc Cas13a Collateral detection 378GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide length 16 AAAACuagauugcuguucuacCas13a Collateral detection 379 GGGGAUUUAGACUACCCCAAAAACGAAGGGGACUguide length 15 AAAACuagauugcuguucua Cas13a Collateral detection 380GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatch 1AAAACAagauugcuguucuaccaaguaauccau Cas13a Collateral detection 381GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatch 3AAAACuaCauugcuguucuaccaaguaauccau Cas13a Collateral detection 382GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatch 5AAAACuagaAugcuguucuaccaaguaauccau Cas13a Collateral detection 383GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatch 7AAAACuagauuCcuguucuaccaaguaauccau Cas13a Collateral detection 384GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatch 9AAAACuagauugcAguucuaccaaguaauccau Cas13a Collateral detection 385GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatchAAAACuagauugcugAucuaccaaguaauccau 11 Cas13a Collateral detection 386GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatchAAAACuagauugcuguuGuaccaaguaauccau 13 Cas13a Collateral detection 387GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatchAAAACuagauugcuguucuUccaaguaauccau 15 Cas13a Collateral detection 388GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatchAAAACuagauugcuguucuacGaaguaauccau 17 Cas13a Collateral detection 389GGGGAUUUAGACUACCCCAAAAACGAAGGGGACU guide Full length mismatchAAAACuagauugcuguucuaccaUguaauccau 19 Target 1 390gggGGCCAGUGAAUUCGAGCUCGGUACCCGGGGAU CCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGU AAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCA UAAAG Target 2 391gggGGCCAGUGAAUUCGAGCUCGGUACCCGGGGAU CCUCUAGAAAUAUGGAUUACUUGGUAGAACAGCAAUGUACUCGACCUGCAGGCAUGCAAGCUUGGCGU AAUCAUGGUCAUAGCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCA UAAAG

SUPPLEMENTARY TABLE 7 Commercial TaqMan probes used in this study.Transcript Product ID (Thermo Fisher) GAPDH 4326317E KRAS Hs00364284_g1PPIB Hs00168719_m1 CXCR4 Hs00607978_s1 ACTB Hs01060665_g1 TINCRHs00542141_m1 PCAT Hs04275836_s1 MALAT1 Hs00273907_s1

SUPPLEMENTARY TABLE 8 Custom TaqMan probes used in this study. GeneProbe sequence Forward Primer Reverse Primer Gluc /56- AAGTTCTGTTTGCCGGCCACGATGTTGAA FAM/ CTGATCT (SEQ GTCT (SEQ ID NO: CCAAGCCCA/ZEN/ID NO: 393 394) CCGAGAACAACGA/ 3IABkFQ/ (SEQ  ID NO: 392

SUPPLEMENTARY TABLE 9 Cas13a orthologs used in this study. Cas13a Cas13aOrganism Accession SEQ ID number abbreviation name number NO: Direct Repeat sequence Cas13a1 LshCas13a Leptotrichia WP_018451595.1 395CCACCCCAATATCGAAGGGGACTAAAAC shahii Cas13a2 LwaCas13a LeptotrichiaWP_021746774.1 396 GATTTAGACTACCCCAAAAACGAAGGGGACTA wadei AAAC Cas13a3LseCas13a Listeria seeligeri WP_012985477.1 397GTAAGAGACTACCTCTATATGAAAGAGGACTA AAAC Cas13a4 LbmCas13a LachnospiraceaeWP_044921188.1 398 GTATTGAGAAAAGCCAGATATAGTTGGCAATA bacterium GAC MA2020Cas13a5 LbnCas13a Lachnospiraceae WP_022785443.1 399GTTGATGAGAAGAGCCCAAGATAGAGGGCAA bacterium TAAC NK4A179 Cas13a6 CamCas13a[Clostridium] WP_031473346.1 400 GTCTATTGCCCTCTATATCGGGCTGTTCTCCAAaminophilum AC DSM 10710 Cas13a7 CgaCas13a Carnobacterium WP_034560163.1401 ATTAAAGACTACCTCTAAATGTAAGAGGACTA gallinarum DSM TAAC 4847 Cas13a8Cga2Cas13a Carnobacterium WP_034563842.1 402AATATAAACTACCTCTAAATGTAAGAGGACTA gallinarum DSM TAAC 4847 Cas13a9Pprcas13a Paludibacter WP_013443710.1 403CTTGTGGATTATCCCAAAATTGAAGGGAACTA propionicigenes CAAC WB4 Cas13a10LweCas13a Listeria WP_036059185.1 404 GATTTAGAGTACCTCAAAATAGAAGAGGTCTAweihenstephanensis AAAC FSL R9-0317 Cas13a11 LbfCas13a ListeriaceaeWP_036091002.1 405 GATTTAGAGTACCTCAAAACAAAAGAGGACTA bacterium FSL AAACM6-0635 (Listeria newyorkensis) Cas13a12 Lwa2cas13a LeptotrichiaWP_021746774.1 406 GATATAGATAACCCCAAAAACGAAGGGATCT wadei F0279 AAAACCas13a13 RcsCas13a Rhodobacter WP_013067728.1 407GCCTCACATCACCGCCAAGACGACGGCGGACT capsulatus SB GAAC 1003 Cas13a14RcrCas13a Rhodobacter WP_023911507.1 408GCCTCACATCACCGCCAAGACGACGGCGGACT capsulatus R121 GAAC Cas13a15 RcdCas13aRhodobacter WP_023911507.1 409 GCCTCACATCACCGCCAAGACGACGGCGGACTcapsulatus GAAC DE442

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed:
 1. A non-naturally occurring or engineered compositioncomprising i) a Cas13a CRISPR-Cas effector protein fused to one or morelocalization signals, or one or more polynucleotide molecules encoding aCas13a CRISPR-Cas effector protein fused to one or more localizationsignals; and ii) one or more nucleic acid components, the one or morenucleic acid components comprising a heterologous guide sequence that iscapable of hybridizing to a target RNA sequence, whereby the effectorprotein forms a complex with the one or more nucleic acid components,the one or more nucleic acid components directs the complex to thetarget RNA sequence in a mammalian target locus and the complex binds tothe target RNA sequence; wherein the Cas13a CRISPR-Cas effector proteinis selected from Cas13a CRISPR-Cas proteins obtained from Eubacteriaceaebacterium CHKCI004, Blautia sp. Marseille-P2398, RNA-binding protein S1Chloroflexus aggregans, Demequina aurantiaca, Thalassospira sp. TSL5-1,SAMN04487830_13920 Pseudobutyrivibrio sp. OR37, SAMN02910398_00008Butyrivibrio sp. YAB3001, Leptotrichia sp. Marseille-P3007, Bacteroidesihuae, SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeriariparia, and Insolitispirillum peregrinum.
 2. The composition of claim1, wherein the Cas 13a effector protein comprises at least two HigherEukaryote and Prokaryotes Nucleotide-binding (HEPN) domain.
 3. Thecomposition of claim 1, wherein at least the one or more nucleic acidcomponents is engineered for use in modifying the mammalian target locusof interest comprising the target RNA sequence.
 4. The composition ofclaim 1, wherein at least one of said one or more localization signalsis a nuclear localization signal (NLS) or a nuclear export signal (NES).5. The composition of claim 1, wherein the one or more polynucleotidemolecules comprise one or more regulatory elements operably configuredto express the polypeptides and/or the nucleic acid component(s).
 6. Thecomposition of claim 5, wherein the one or more regulatory elementscomprise one or more promoters or one or more inducible promoters. 7.The composition of claim 1, wherein the Cas13a CRISPR-Cas effectorprotein is associated with one or more functional domains.
 8. Thecomposition of claim 1, wherein the effector protein contains one ormore mutations within a HEPN Domain corresponding to R597A, H602A,R1278A, and/or H1283A of Leptotrichia shahii, or the corresponding aminoacids in a Cas13a ortholog.
 9. The composition of claim 1, wherein thenucleic acid component comprises a dual direct repeat sequence.
 10. Thecomposition of claim 1, wherein the one or more polynucleotide moleculesare comprised within one or more vectors.
 11. The composition of claim10, wherein the one or more vectors are viral vectors.
 12. Thecomposition of claim 11, wherein the one or more viral vectors compriseone or more retroviral, lentiviral, adenoviral, adeno-associated orherpes simplex viral vectors.
 13. The composition of claim 1, whereinthe assembled complex is comprised in a delivery system.
 14. Thecomposition of claim 1, wherein the non-naturally occurring orengineered composition is delivered via a delivery vehicle comprisingliposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun or oneor more viral vector(s).
 15. A eukaryotic cell engineered to comprise orexpress, the composition or a component thereof of: a non-naturallyoccurring or engineered composition comprising i) a Cas13a CRISPR-Caseffector protein or one or more polynucleotide molecules encoding Cas13aCRISPR-Cas effector protein; and ii) one or more nucleic acidcomponents, the one or more nucleic acid components comprising aheterologous guide sequence that is capable of hybridizing to a targetRNA sequence, whereby the effector protein forms a complex with the oneor more nucleic acid components, the one or more nucleic acid componentsdirects the complex to the target RNA sequence in a mammalian targetlocus and the complex binds to the target RNA sequence; wherein theCas13a CRISPR-Cas effector protein is selected from Cas13a CRISPR-Casproteins obtained from Eubacteriaceae bacterium CHKCI004, Blautia sp.Marseille-P2398, RNA-binding protein S1 Chloroflexus aggregans,Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920Pseudobutyrivibrio sp. OR37, SAMN02910398_00008 Butyrivibrio sp.YAB3001, Leptotrichia sp. Marseille-P3007, Bacteroides ihuae,SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeriariparia, and Insolitispirillum peregrinum.
 16. The cell according toclaim 15, wherein the cell comprises a mammalian cell.
 17. Amulticellular organism comprising one or more cells according to claim15.
 18. A plant or animal model comprising one or more cells accordingto claim 15, wherein said one or more cells express the composition or acomponent thereof.
 19. A method of modifying a target sequence, themethod comprising delivering to a locus of interest comprising themammalian target sequence a non-naturally occurring or engineeredcomposition comprising i) a Cas13a CRISPR-Cas effector protein fused toone or more localization signals, or one or more polynucleotidemolecules encoding a Cas13a CRISPR-Cas effector protein fused to one ormore localization signals; and ii) one or more nucleic acid components,the one or more nucleic acid components comprising a heterologous guidesequence that is capable of hybridizing to a target RNA sequence,whereby the effector protein forms a complex with the one or morenucleic acid components, the one or more nucleic acid components directsthe complex to the target RNA sequence in a mammalian target locus andthe complex binds to the target RNA sequence; wherein the Cas13aCRISPR-Cas effector protein is selected from Cas13a CRISPR-Cas proteinsobtained from Eubacteriaceae bacterium CHKCI004, Blautia sp.Marseille-P2398, RNA-binding protein S1 Chloroflexus aggregans,Demequina aurantiaca, Thalassospira sp. TSL5-1, SAMN04487830_13920Pseudobutyrivibrio sp. OR37, SAMN02910398_00008 Butyrivibrio sp.YAB3001, Leptotrichia sp. Marseille-P3007, Bacteroides ihuae,SAMN05216357_1045 Porphyromonadaceae bacterium KH3CP3RA, Listeriariparia, Insolitispirillum peregrinum, Herbinix hemicellulosilityca, andLeptrotrichia buccalis C-1013-b.
 20. The method of claim 19, wherein themodification of the target sequence comprises a strand break.
 21. Themethod of claim 19, wherein the Cas13a CRISPR-Cas effector protein isassociated with one or more functional domains.
 22. The method of claim21, wherein the one or more functional domains modifies transcription ortranslation of the target locus.
 23. The method of claim 21, wherein theeffector protein contains one or more mutations within an HEPN Domaincorresponding to R597A, H602A, R1278A, and/or H1283A H1283A ofLeptotrichia shahii, or the corresponding amino acids in a Cas13aortholog, whereby the complex can deliver an epigenetic modifier or atranscriptional or translational activation or repression signal. 24.The method of claim 19, wherein at least one of said one or more saidlocalization signals is a nuclear localization signal (NLS) or a nuclearexport signal (NES).
 25. The method of claim 19, wherein the one or morepolynucleotide molecules comprise one or more regulatory elementsoperably configured to express the polypeptides and/or the nucleic acidcomponent(s).
 26. The composition of claim 25, wherein the one or moreregulatory elements comprise one or more promoters or one or moreinducible promoters.